Compositions and methods for treating cellular response to injury and other proliferating cell disorders regulated by hyaladherin and hyalurons

ABSTRACT

The present invention provides compositions and methods for treating a tissue disorder associated with a response-to-injury process or proliferating cells in a mammal. These tissue disorders are associated with a novel cellular phenotype designated as “transition cells” which are described herein. This cellular phenotype is characterized in having an activated erk kinase signaling activity, a stimulated AP-1 binding activity, and at least one characteristic selected from the group consisting of: (a) increased podosome formation, (b) increased flux of intracellular or extracellular hyaluronans or hyaladherins, (c) increased expression of a hyaladherin, (d) an inability to form focal adhesions, (e) increased metalloproteinase activity, and (f) increased expression of a hyaladherin. Example tissue disorders include fibrosis, inflammation, degeneration and invasive disorders such as occur in cancerous cells. The methods provided herein include administering to the mammal, an effective amount of a composition that alters the activity of transition molecules within a cell Transition molecules are shown to be comprised of hyaladherins, hyaluronans and associated molecules that regulate the transitional phenotype. A novel cell culture comprising transition cells is also provided, as well as compositions comprising particular peptides, polypeptides, and antibodies that affect the transitional phenotype.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 09/541,5227 filed Apr. 3, 2000; which application claims thepriority to U.S. Provisional Patent Application No. 60/127,457, filedApr. 1, 1999, all of which applications are incorporated by reference intheir entirety.

TECHNICAL FIELD

The present invention relates generally to the understanding, diagnosisand treatment of a wide variety of diseases, and more specifically, tocompositions and methods for treating cellular response to injury and/orthe abnormal proliferation of cells.

BACKGROUND OF THE INVENTION

The normal cell in normal tissue is confined to a narrow range offunction and structure regulated by its differentiation state, geneticprogram of metabolism, tissue specialization, by constraints induced byneighboring cells, the extracellular matrix and availability ofexogenous factors and metabolic substrates. Cells are able to handlenormal physiological demands (homeostasis) or adapt to excessivephysiological stresses and pathological stimuli by altering their steadystate to preserve cell viability and function. If the adaptive responsesto stimuli are exceeded, then a sequence of cellular events follow thattransform normal cells to injured or diseased cells in an attempt toremodel local tissue. This process can lead to a number of diseasesdriven by enhanced cell proliferation, migration and invasion,production of matrix metalloproteinases, infiltration of inflammatorycells, tissue destruction and dysfunctional tissue remodeling.Regardless of the etiology, such disease processes are commonly found ininflammatory diseases (such as arthritis, multiple sclerosis, psoriasis,inflammatory bowel diseases, diabetes), proliferative diseases (such ascancer and metastases), degenerative diseases (such as osteoarthritis,osteoporosis, Alzheimer's. Parkinson's) and injuries caused by wounds orburns.

Current medical approaches to treat or prevent such diseases typicallyinvolve the use of reagents that attempt to block mechanisms affectingcell proliferation, cell migration, or the production of enzymes orgrowth factors. However, because such reagents are not specific todiseased cells and current practices do not et allow targeting of thesereagents specifically to sites of disease, such a therapeutic approachis typically toxic to the host if used for any length of time or at highdosages as may be required to treat or prevent the disease. Thistoxicity of current reagents is a severe limitation to the efficacy ofcurrent medical treatments.

The present invention discloses a sequence of cellular transition statesthat are involved in the transformation of normal cells to diseasedcells that is characteristic to all cell types, and thus, all tissues.Transitory molecules produced during the early phases of disease whichare responsible for the transition of cells from normal to diseasedstate are described, as well as the use of such molecules in thediagnosis, treatment and/or prevention of a wide variety of diseases isprovided.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides compositions and methodsfor treating a tissue disorder associated with a response-to-injuryprocess, or proliferating cells in a mammal. The methods includeadministering to the mammal an effective amount of a composition thatalters the activity of transition molecules within a cell Transitionmolecules are comprised of hyaladherins, hyalauronans or moleculesregulated by an amount of intracellular or extracellular hyaladherins orhyalauronans. The activity of hyaladherins and hyalauronans are shown tointeract with regulatory processes associated with a response to injuryand/or proliferative/invasive cell types.

Within one aspect of the present invention, methods are provided fortreating a wide variety of inflammatory or proliferative diseases,comprising the step of administering to a warm-blooded animal (e.g. ahuman, horse, cow, sheep, dog, cat, or mouse) a compound to treat theinflammatory or proliferative disease.

A wide variety of compounds may be utilized within this regard,including for example (a) a polypeptide comprising the amino acidsequence BX7B (SEQ ID NO:28) which binds HA; phage display selectedpeptides that bind HA such as polypeptides comprising P-15 (Sequence IDNo. 70). P-16 (Sequence ID No. 26): P-3′ (Sequence ID no. 71); andGAHWQFNALTVR (Sequence ID No. 72); (b) an antibody which binds any oneof domains D1, D2, D3, D4, or. D5 of RHAMM (e.g. an anti-TAM antibody);(c) a polypeptide fragment which encodes a D1, D2, D3, D4, or, D5 domainof RHAMM: and (d) a gene deliver, vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c). In oneembodiment, the polypeptide can be (a) a first peptide comprised of ahyaluronic acid-binding domain; (b) a hyaladherin polypeptide; (c) asecond peptide comprised of a domain from a hyaladherin polypeptide; (d)a hyaladherin-binding polypeptide; (e) a third peptide comprised of ahyaladherin binding domain. Also provided are antibodies which binds toa peptide or polypeptide of (a)-(d); and/or vectors (e.g. gene deliveryvectors described below) that expresses a gene encoding a polypeptide asdescribed above or herein. More particular embodiments include methodswhere a hyaladherin is administered comprised of a RHAMM polypeptide. Ina particular embodiment, a first peptide is administered comprised of asequence selected from the group consisting of SEQ. ID NO: 1-10. Inanother embodiment, a second peptide is administered comprised of asequence selected from the group consisting of SEQ. ID NO: 11-20. Inanother embodiment, a hyaladherin-binding polypeptide comprised of SEQ.ID NO: 21. In still other embodiments, an antibody is administered thatbinds to a peptide comprised of SEQ. ID NO: 11-20 or an antibody isadministered that binds to a RHAMM polypeptide. In a more generalembodiment, at least one hyaladherin is administered that is a RHAMMpolypeptide.

Within other embodiments, the compound to be delivered is a polypeptidecomprising less than an entire RHAMM polypeptide (i.e., less than 95 kDor 73 kD molecular weight), and containing at least a portion of RHAMMdomains D1 (e.g., aa 1-164 of human RHAMM). D2 (the leucine zipperdomain, e.g. aa 195-122 of human RHAMM), D3 (the “TAM domain”, e.g. aa219-240 of human RHAMM). D4 (the repeat or “R” domain—e.g., aa. 442-546of mouse and aa. 442-463 of human RHAMM), or. D5 (two HA bindingdomains—e.g., aa. 721-730 and 742-752 of mouse RHAMM, and aa. 6′)-645and aa. 657-666 RHAMM). Equivalent domains in other species may bedetermined by comparison with the human sequence (see e.g. FIG. 50: SEQID Nos. 47 and 48). Within further embodiments, antibodies are providedwhich bind to any one of the above RHAMM domains (i.e., domains D1, D2,D2, D4, or, D5). Examples include antibodies to SEQ ID Nos. 14, 17, 23,24, 26 and 27.

A wide variety of inflammatory or proliferative diseases may be treatedwith the aforementioned compounds, including for example, inflammatoryor proliferative neurological diseases such as Parkinsons or Alzheimer'sdisease, arthritic diseases (e.g. rheumatoid arthritis andosteoarthritis), diseases associated with demylination of the nervesheath (e.g., multiple sclerosis), inflammatory dermatosis (e.g.,psoriasis), inflammatory bowel diseases, a variety of wounds (e.g.surgical excisions adhesions, scars, and cheloids, and skin ulcers),stenosis and/or restenosis (as well as proliferative, inflammatoryresponses or fibrotic response associated with medical implants, such aship implants, vascular wraps, catethers, and the like), cancer and othermalignant diseases, kidney fibrosis, inflammatory lung diseases (e.g.,emphysema, asthma, and cystic fibrosis), obesity and obesity-relateddiseases (including for example, diabetes), lupus, tissuetransplantation (e.g., skin grafts) and cardiovascular diseases (e.g.,atherosclerosis, and stroke).

Within the context of the present invention, it should be noted that theabove-noted diseases are deemed to be “treated” if the typical diseasecourse is altered, slowed, inhibited, or prevented in a statisticallysignificant manner, for at least one symptom or sign of the disease.

In related embodiments, the invention provides a composition fortreating a tissue disorder associated with a response-to-injury processor proliferating cells in a mammal comprising the compositions describedin the aforementioned methods of treating.

In a different aspect, the invention provides cell cultures which arecomprised of a transition cells, wherein the transition cells include anactivated erk kinase signaling activity, a stimulated AP-1 bindingactivity and at least one characteristic selected from the groupconsisting of: (a) increased podosome formation. (b) increased flux .ofintracellular or extracellular hyalauronans or hyaladherins. (c)increased expression of a hyaladherin, (d) an inability to form focaladhesions. (e) increased metalloproteinase activity, and (f) increasedexpression of hyaladherin; wherein measurements of activity or amountsare relative to a normal quiescent cell. In more specific embodiments,the cell culture is comprised of cells transfected with a nucleic acidthat overexpresses a hyaladherin. In a still more specific embodiment,the cell culture overexpresses a RHAMM polypeptide. In a related aspect,the invention provides embodiments of an animal cell culture wherein theactivated erk signaling activity is less than the activated erksignaling activity displayed by normal quiescent cells in response to agrowth factor stimulation, wherein the growth factor stimulation isselected from the group consisting of: IL-1 stimulation, PDGFstimulation, and NTF α stimulation.

In still another aspect, the invention provides methods of identifying acomposition for treating a tissue disorder associated with aresponse-to-injury process or proliferating cells in a mammalcomprising, (a) treating the cell culture as described above with acandidate composition; (b) scoring for the ability of the candidatecomposition to inhibit podosome formation in comparison to an untreatedculture containing transition cells as described above; and (c)selecting the composition that inhibits podosome formation. Oneembodiment provides a method where scoring for the ability to inhibitpodosome formation comprises testing the treated cell culture for areduced ability to release a protease that degrades a cellular matrixcomponent. In a more particular embodiment of the aforementioned method,the protease is a fibronectin degrading protease that releases a CS-1sequence from a fibronectin matrix, and the scoring comprises testingthe ability of the treated cells to bind an antibody specific for theCS-1 sequence.

In a different aspect, the invention provides peptide compositions thatbind a hyalauronan comprising a peptide of the sequence BX7B (SEQ IDNO:28) wherein B is a basic amino acid and X7 is a sequence of aboutseven residues selected from any amino acid other than an acidic aminoacid, wherein the peptide forms an alpha helix and each occurrence of Bis oriented on the same side of the alpha helix, and with the provisothat the peptide does not consist of the sequences BBXXBBBXXBB,KQKIKHVVKLK, KLKSQLVKRK, RYPISRPRKR, KNGRYSISR, RDGTRYVQKGEYR,RRRCGQKKK, RGTRSGSTR, RRRKKIQGRSKR, RKSYGKYQGR, KVGKSPPVR, KTFGKMKPR,RIKWSRVSK, KRTMRPTRR, KVGKSPPVR, or HREARSGKYK (SEQ ID Nos. 29-44respectively).

In a related aspect, the invention provides peptide compositions thatbind a hyalauronan comprising a peptide of the sequence BX7B (SEQ IDNO:28) wherein B is a basic amino acid and X7 is a sequence of sevenresidues selected from amino acids other than acidic amino acids,wherein the peptide satisfies one of the following (sets ofrequirements: (a) the amino acid residue at position 4, 5 or 6 is K, H,R, A, C, G, S, or T, (e.g., P-peptide (RSHKTRSHH; SEQ ID NO:61) andR-peptide (RGGGRGRRR: SEQ ID NO:27)); (b) the amino acid residue atposition 4 or 5 is K. H. R. A. C. S, or T and the amino acid residue atposition 6 is A, P, F, W, M, I, V, or L; (c) the amino acid residues atpositions 2 and 6 are I, L, or V, and the amino acid residue at position5 is K, H, R, Q, or N, (e.g., fibromodulin (KVGRKVFSK; SEQ ID NO:64),CD40 receptor associated factor-1 (KCSVQTLLR; SEQ ID NO:65), probable Gprotein coupled receptor (RTHLKHVLR; SEQ ID NO:66), leukoctye derivedchemotaxin-2 (KNAINNGVR; SEQ ID NO:67), laminin gamma-2 (KGQINNSIK; SEQID NO:68)); (d) the amino acid residues at position 2 and 6 are I, L, orV, the amino acid residue at position 4 is N, C, Q, G, S, or T, theamino acid residue at position 5 is R, N, C, Q, H, or K, the amino acidresidue at position 8 is R, N, C, Q, G, H, K, S, or T, and the aminoacid residues at positions 3 and 7 are any residue except D, E, P, W, Y,M, or F; (e) the amino acid residues at positions 2 and 6 are A, I, L,or V, the amino acid residue at position 3 is anything except D, E, P,W, Y, M, or F, the amino acid residue at position 5 is R, N, Q, H, or K,the amino acid residue at position 7 is H, R, or K, (e.g., cdc37(RVRGRAKLR; SEQ ID NO: 69)); or (f) the amino acid residues at positions2 and 6 are I, L, or V, the amino acid residue at position 3 is anyresidue except D, E, P, W, Y, M, or F, the amino acid residue atposition 4 is K, R, or H, the amino acid residue at position 5 is R, N,Q, H, or K, and the amino acid residue at position 7 is H, R, or K. Theinvention also provides peptide compositions comprising a peptide thatis a part of any peptide that meets the requirements of set (b)described above as long as the peptide has no acidic residues and alsosatisfies the requirements of set (b).

In yet another related aspect, peptide compositions are provided thatbinds a hyalauronan or block podosome formation in a cell, wherein thepeptide is selected from group consisting of: (H4-5)B3 (SEQ. ID. NO: 1);(H4-5)BXBBXB (SEQ. ID. NO: 2); (H4-5)BXBXBBB (SEQ. ID. NO: 3);(H4-5)BXBBB (SEQ. ID. NO: 4); and (H4-5)BXBB (SEQ. ID. NO: 5). In theseembodiments B is lysine (K) or arginine (R), X is a hydrophobic orneutral amino acid selected from the group consisting of (I, L, V, Q, S)and H4-5 is sequence of 4 to 5 amino acids selected to form an alphahelix. In more particular embodiments of this aspect, the peptide isselected from the group consisting of: MMTVLKR (SEQ. ID. NO: 6);MMTVLKVKRLR (SEQ. ID. NO: 7): MMTVLKVKVKRK (SEQ. ID. NO: 8); MMTVLKVRKR(SEQ. ID. NO: 9); and MMTVLKVRK (SEQ. ID. NO: 10).

In yet a different aspect, the invention provides methods of identifyinga peptide or polypeptide composition for treating a tissue disorderassociated with a response-to-injury process or proliferating cells in amammal comprising the steps of: (a) selecting a sequence from a databasescreened for sequences comprising a peptide of the sequence BX7B (SEQ IDNO:28) wherein B is a basic amino acid and X7 is a sequence of aboutseven residues selected from any amino acid other than an acidic aminoacid, wherein the peptide forms an alpha helix and each occurrence of Bis oriented on the same side of the alpha helix, (b) preparing acomposition comprised of the selected sequence; and (c) testing theprepared composition for the ability to inhibit podosome formationaccording to other methods taught by this invention, and with theproviso that the peptide in (a) does not consist of the sequencesBBXXBBBXXBB, KQKIKHVVKLK, KLKSQLVKRK. RYPISRPRKR, KNGRYSISR,RDGTRYVQKGEYR, RRRCGQKKK, RGTRSGSTR, RRRKKIQGRSKR, RKSYGKYQGR,KVGKSPPVR, KTFGKMKPR, RIKWSRVSK, KRTMRPTRR, KVGKSPPVR, or HREARSGKYK(SEQ ID Nos. 29-44 respectively). Within certain embodiments thepeptides provided herein can be less than 250, 200, 150, 100, 75, 50,or, 25 amino acids in length.

In a related aspect, the invention provides methods of identifying apeptide or polypeptide composition for treating a tissue disorderassociated with a response-to-injury process or proliferating cells in amammal comprising the steps of: (a) forming an expression librarycomprised of cloned sequences expressed by a cell during a transitionstage response; (b) screening the expression library for sequencesencoding a peptide or polypeptide that binds a hyaladherin that isstimulated in cells during the transition stage: (c) preparing a peptideor polypeptide encoded by the hyaladherin binding sequences; and (d)testing the peptide or polypeptide for the ability to affect at leastone activity associated with transition stage cells, wherein theactivity is selected from the group consisting of: increased erk kinasesignal activation, podosome formation, metalloproteinase expression,flux of intracellular or extracellular hyaluronans or hyaladherins,expression of a hyaladherin, and inability to form focal adhesions. Inone embodiment of this method, the expression vector is a two hybridphage display system, the hyaladherin is RHAMM and the testing is forthe ability to inhibit podosome formation and inhibition of erk kinasesignaling activation.

In a further aspect of the present invention polypeptides are providedwhich comprises or consist only of any one of SEQ ID Nos 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60,61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or, 72. When the polypeptidecomprises additional sequences, the polypeptide may, in certainembodiments, be less than 50 kd or 20 kd of molecular weight. In otherembodiments, the polypeptide may be less than 60, 50, 40, 30, or, 20amino acids in length. Also provided are such polypeptides that arepurified (e.g., free of cells), sterilized, and/or suitable forpharmaceutical use.

In yet other aspects of the present invention antibodies are providedwhich bind to any one of the peptides provided herein, including forexample, a polypeptide comprising or consisting only of any one of SEQID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 56, 57, 58, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or,72. Also provided are antibodies such as noted above, which are purified(e.g., free of cells), sterilized, and/or suitable for pharmaceuticaluse (e.g. for use in one of the methods described herein).

In still another aspect, the invention provides methods for detectinghyalauronic acid in a sample comprising the steps of: (a) incubating thesample with a hyalauronic acid binding peptide comprising a sequenceselected from the group consisting of SEQ. ID NO: 1-10; and (b)detecting an amount of a complex formed between hyalauronic acid and thehyalauronic acid binding peptide. In one embodiment, the detectingemploys an antibody that specifically binds to the hyalauronic acidbinding peptide.

In a related aspect, the invention provides methods of detecting amolecule that binds to a RHAMM polypeptide in a sample comprising thesteps of (a) incubating the sample with the RHAMM polypeptide and with aRHAMM-binding polypeptide comprised of SEQ. ID NO: 21; and (b) detectingan amount of a complex formed between the sample and the RHAMMpolypeptide by scoring for reduced binding between the RHAMM polypeptideand the RHAMM-binding polypeptide. In one embodiment, detecting employsan antibody that specifically binds to the RHAMM-binding polypeptide.

Yet another embodiment of the invention includes compositions fortreating a tissue disorder associated with a response-to-injury processor proliferating cells in a mammal, wherein the composition is comprisedof a hyaluronan that binds to a hyaladherin that regulates a transitionstage cell associated with the response to injury or proliferating celldisorder. In a related aspect, the invention provides a method fortreating a tissue disorder associated with a response-to-injury processor proliferating cells in a mammal comprising, administering to themammal the aforementioned hyaluronan.

Within further aspects of the present invention, vaccinating agents areprovided for preventing a tissue disorder associated with aresponse-to-injury process or proliferating cells in a mammal whereinthe vaccinating agent includes an antigen comprised of one of theaforementioned polypeptides, peptides or hyaluronans. Generally, withinthis aspect, the vaccinating agent is administered in a dosage and/ortherapeutic regimen sufficient to generate an immune response againstthe selected antigen, and to prevent the tissue disorder orproliferation of cells.

These and other aspects of the present invention will become evidentupon reference to the following detailed description and attacheddrawings. In addition, various references are set forth herein whichdescribe in more detail certain procedures or compositions (e.g.,plasmids, etc.) and are therefore incorporated by reference in theirentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the impact of the AP-1 pathway ondisease.

FIG. 2 is a schematic illustration of cell activation in response to avariety of factors, and this impact on disease pathways.

FIG. 3 depicts cells transition from a normal cell to a diseased cell.

FIG. 4 shows immunofluorescence micrographs and phosphoprotein assayswhich indicate a requirement for cell adhesion for activation of theerk1 kinase signaling.

FIG. 5 schematically illustrates the involvement of RHAMM as atransitional molecule.

FIGS. 6A and 6B are two blots which show erk activity.

FIGS. 7A and 7B show the increased expression levels of e-fos, c-jun,junB genes by overexpression of RHAMM.

FIG. 8 shows the increased expression levels of c-fos and c-jun gene incells overexpressing RHAMM regardless whether grown on PL or FN.

FIGS. 9A and 9B are northern blots probed with gelatinase B,stromelysin, timp-1, and GAPDH CDNAS.

FIG. 10 is a blot which shows that LR21 cells overexpressing RHAMMv4 arerestricted in the extent to which proinflammatory cytokines can activateerk kinase.

FIGS. 11A and 11B are northern analysis of IL-1 and TNF-alpha inductionof c-fos.

FIGS. 12A and 12B are photographs of LR21 cells, showing, in 12A, theformation of discrete focal adhesions.

FIGS. 13A and 13B are photos, and graphs (respectively), which shortthat overexpression of RHAMM results in podosome formation.

FIGS. 14A, 14B, and 14C are graphs which illustrate the relationship ofRHAM, erk activity and podosome formation. FIGS. 14D, 14E, and 14F arephotos which supplement this data.

FIG. 15A is a bar graph which shows a comparison of RHAMM expression ata cell surface. FIG. 15B provides the sequence of various RHAMM peptides(SEQ ID Nos. 14-20).

FIGS. 16A and 16B are photographs showing cells treated with peptides.

FIGS. 16C and 16D are bar graphs quantifying the effect.

FIGS. 17A and 17B are photographs showing podosome formation undervarious conditions.

FIG. 18 is two photographs showing MDA-231 cells trated with hyaluronantogether with anti-RHAMM antibody.

FIG. 19 is a chart that compares rate of locomotion for various celllines and various substrates.

FIGS. 20A and 20B show that anti-RHAMM antibodies inhibit the ability ofMDA231 cells to invade in vitro.

FIGS. 21A, 21B and 21C show RHAMM binding to fibronectin being blockedby selected antibodies.

FIG. 22 is a bar graph which shows that MDA231 cells expressing RHAMMhave a high degree of motility.

FIGS. 23A and 23B are micrographs which show podosome formation invarious cells.

FIGS. 24A and 24B are a graph, and a blot, respectively, which show theeffects of exon 4 antibody and LZP on the formation of podosomes.

FIGS. 25A-D show that both v4 and v5 forms of RHAMM associated with erk1in vivo and in vitro, but that only the short form stongly activates theerk kinase cascade.

FIGS. 26A, 26B and 26C illustrate that HA binding peptides (SEQ ID Nos.28,56-58) including artificial mimics are able to block cell motility.

FIG. 27 is a bar graph which shows that treatment of injured cells withP-peptide (CSTMMSRSHKTRSHHV-Seq. ID. No. 26) inhibits migration of HFFcells.

FIG. 28 is a bar graph which shows velocity of cells after addition ofpeptide aa423-432 (Sequence ID No. 24).

FIG. 29 is two bar graphs which, show MMP release from knockoutfibroblasts compared to normal ones (on fibronectin vs. cell cultureplastic).

FIG. 30 is a bar graph and blots which show that erk phosphorylation isinfluenced by RHAMM expression.

FIG. 31 is a bar graph which shows knockout fibroblasts have decreasedmotility compared to wild-type fibroblasts.

FIGS. 32 a, 32 b, 32 c and 32 d are photographs of bleomycin-inducedlung fibrosis.

FIG. 33 is a bar graph which illustrates that a significant increase inmotility of macrophages from both bleomycin and saline-treated animals.

FIG. 34 is a bar graph which illustrates the motility of BAL cells fourdays after injury in response to administration of RHAMM peptides.

FIG. 35 is a bar graph, and a blot which shows the ability of HA bindingpeptides to inhibit firbosis.

FIGS. 36 a-f are a series of photographs from a histological analysis oflung tissue.

FIG. 37 is a table which shows the percentage of cells with variousisoforms of RHAMM, from a variety of RA patients.

FIGS. 38A-F are a series of photographs of stained RA tissue.

FIG. 39 is a graph which shows attenuation of clinical signs of MS aftertreatment.

FIGS. 40A and 40B are bar graphs which show that collagen productionfollowing treatment with P-peptide.

FIG. 41 shows that P-peptide reduces infiltration of macrophages intothe site of a wound.

FIG. 42 is a bar graph which compares glucosamine activity aftertreatment of various peptides.

FIGS. 43A and 43B illustrate expression of RHAMM isoforms from variouscell lines.

FIGS. 44A (bar graphs) and 44B (a blot) illustrate random cell motilityand matrigel cell invasion utilizing various peptides.

FIG. 45 is a graph which shows weight change in transgenic mice.

FIGS. 46A and 46B show that RHAMM is most highly expressed in the mostinvasive lung cancer cell lines.

FIGS. 47A and 47B show that RHAMM is most highly expressed in high gradeor invasive astnocytomas.

FIG. 48A is a schemata showing domains of various RHAMM polypeptidesrequired for podosome formation and activation of erk kinase signalingand FIG. 48B is a protein gel showing that intracellular RHAMMv4 bindsto ERK kinase.

FIG. 49 shows (A) a partial amino acid (SEQ ID NO:46) and nucleotidesequence (SEQ ID NO:45) of a RHAMM binding protein (RABP) isolated usinga phage two hybrid system; (B) a Northern blot of RABP expression intransitional cells; (C) a Western blot of transitional cell lysateindicating that RABP is a 60 kDa protein; and (D) a FACS analysisillustrating that RABP is present on the cell surface.

FIG. 50 depicts the human and murine sequence of RHAMM (SEQ ID Nos. 47and 48 respectively).

FIG. 51 is a bar graph that depicts the incidence of abnormal bloodglucose levels in NOD mice.

FIG. 52 is a bar graph that depicts the incidence of abnormal urineglucose level in NOD mice.

FIG. 53 is a graph that indicates the effect of P-16 peptide on waterconsumption in NOD mice.

FIG. 54 is a graph that indicates the effect of P-16 peptide on kidneyweight in NOD mice.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Prior to setting forth the invention, it may be helpful to anunderstanding, thereof to first set forth definitions of certain termsthat will be used hereinafter.

“Expression vector” and “expression cassette” refers to an assemblywhich is capable of directing the expression of a sequence or gene ofinterest. The nucleic acid expression vector must include a promoterwhich, when transcribed, is operably linked to the sequence(s) orgene(s) of interest, as well as a polyadenylation sequence. Withincertain embodiments of the invention, the expression vectors describedherein may be contained within a plasmid construct. In addition to thecomponents of the nucleic acid expression vector, the plasmid constructmay also include a bacterial origin of replication, one or moreselectable markers, a signal which allows the plasmid construct to existas single-stranded DNA (e.g., a M13 origin of replication), a multiplecloning site, and a “mammalian” origin of replication (e.g., an SV40 oradenovirus origin of replication).

As used herein, “nucleic acid” or “nucleic acid molecule” refers to anyof deoxyribonucleic acid (DNA), ribonucleic acid (RNA),oligonucleotides, fragments generated by the polymerase chain reaction(PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acids can becomposed of monomers that are naturally-occurring nucleotides (such asdeoxyribonucleotides and ribonucleotides), or analogs ofnaturally-occurring nucleotides (e.g. α-enantiomeric forms ofnaturally-occurring nucleotides), or a combination of both. Modifiednucleotides can have modifications in sugar moieties and/or inpyrimidine or purine base moieties. Sugar modifications include, forexample, replacement of one or more hydroxyl groups with halogens, alkylgroups, amines, and azido groups, or sugars can be functionalized asethers or esters. Moreover, the entire sugar moiety can be replaced withsterically and electronically similar structures, such as aza-sugars andcarbocyclic sugar analogs. Examples of modifications in a base moietyinclude alkylated purines and pyrimidines, acylated purines orpyrimidines, or other well-known heterocyclic substitutes. Nucleic acidmonomers can be linked by phosphodiester bonds or analogs of suchlinkages. Analogs of phosphodiester linkages include phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the like.The term “nucleic acid” also includes so-called “peptide nucleic acids.”which comprise naturally-occurring or modified nucleic acid basesattached to a polyamide backbone. Nucleic acids can be either singlestranded or double stranded.

An “isolated nucleic acid molecule” is a nucleic acid molecule that isnot integrated in the genomic DNA of an organism. For example, a DNAmolecule that encodes a RHAMM binding protein that has been separatedfrom the genomic DNA of a eukaryotic cell is an isolated DNA molecule.Another example of an isolated nucleic acid molecule is achemically-synthesized nucleic acid molecule that is not integrated inthe genome of an organism.

An “isolated polypeptide” is a polypeptide that is essentially free fromcontaminating cellular components, such as carbohydrate, lipid, or otherproteinaceous impurities associated with the polypeptide in nature. Thata particular protein preparation contains an isolated polypeptide can beshown by the appearance of a single band following sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis of the proteinpreparation and Coomassie Brilliant Blue staining of the gel.

“Gene delivery vehicle” refers to a construct which is capable ofdelivering, and, within preferred embodiments expressing, one or moregene(s) or sequence(s) of interest in a host cell. Representativeexamples of such vehicles include viral vectors, nucleic acid expressionvectors, naked DNA, and certain eukaryotic cells (e.g., producer cells).

A “ribozyme” is a nucleic acid molecule that contains a catalyticcenter. The term includes RNA enzymes, self-splicing RNAs, self-cleavingRNAs, and nucleic acid molecules that perform these catalytic functions.A nucleic acid molecule that encodes a ribozyme is termed a “ribozymegene.”

“Treatment” refers to both therapeutic treatment and prophylactic orpreventative measures. Those in need of treatment include those alreadywith the disorder as well as those prone to have the disorder or thosein which the disorder is to be prevented.

“Mammal” for purposes of treatment refers to any animal classified as amammal, including humans, domestic and farm animals, and zoo, sports, orpet animals, such as dogs, horses, cats, cows, etc. Preferably, themammal herein is human.

“Antibodies” (Abs) and “immunoglobulins” (Igs) are glycoproteins havingthe same structural characteristics. While antibodies exhibit bindingspecificity to a specific antigen, immunoglobulins include bothantibodies and other antibody-like molecules for which antigenspecificity has not been defined. Polypeptides of the latter kind are,for example, produced at low levels by the lymph system and at increasedlevels by myelomas.

“Native antibodies and immunoglobulins” are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (V.sub.H) followed by a number of constant domains. Eachlight chain has a variable domain at one end (V.sub.L) and a constantdomain at its other end: the constant domain of the light chain isaligned with the first constant domain of the heavy chain, and the lightchain variable domain is aligned with the variable domain of the heavychain. Particular amino acid residues are believed to form an interfacebetween the light- and heavy-chain variable domains (Clothia et al., J.Mol. Biol. 186:651 (1985); Novotny and Haber, Proc. Natl. Acad. Sci.U.S.A. 82:4592 (1985)).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called complementarity-determining regions (CDRs) orhypervariable regions both in the light-chain and the heavy-chainvariable domains. The more highly conserved portions of variable domainsare called the framework (FR). The variable domains of native heavy andlight chains each comprise four FR regions, largely adopting a.beta.-sheet configuration, connected by three CDRs, which form loopsconnecting, and in some cases forming part of, the .beta.-sheetstructure. The CDRs in each chain are held together in close proximityby the FR regions and, with the CDRs from the other chain, contribute tothe formation of the antigen-binding site of antibodies (see Kabat etal. Sequences of Proteins of Immunological Interest. Fifth Edition.National Institute of Health, Bethesda, Md. (1991)). The constantdomains are not involved directly in binding an antibody to an antigen,but exhibit various effector functions, such as participation of theantibody in antibody-dependent cellular toxicity.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V.sub.H -V.sub.L dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H)-1) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxy terminus of the heavy chain C_(H)-1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa (.kappa.) and lambda (.lambda.), based on the amino acid sequencesof their constant domains.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these can be further divided into subclasses(isotypes), e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3. IgG.sub.4, IgA.sub.1,and IgA.sub.2. The heavy-chain constant domains that correspond to thedifferent classes of immunoglobulins are called .alpha., .delta.,.epsilon., .gamma., and .mu., respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

The term “monoclonal antibody” (mAb) as used herein refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed against a single antigenic site. Furthermore, in contrastto conventional (polyclonal) antibody preparations which typicallyinclude different antibodies directed against different determinants(epitopes), each mAb is directed against a single determinant on theantigen. In addition to their specificity, the monoclonal antibodies areadvantageous in that they can be synthesized by hebridoma culture,uncontaminated by other immunoglobulins. Thus, the modifier “monoclonal”indicates the character of the antibody as being obtained from asubstantially homogeneous population of antibodies, and is not to beconstrued as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention can be made by the hybridoma method firstdescribed by Kohler and Milstein, Nature 256:495 (1975), or can be madeby recombinant DNA methods (Cabilly et al., supra).

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (Cabilly et al., supra;Morrison et al., Proc. Natl. Acad. Sci. U.S.A. 81:6851 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are specificchimeric immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv. Fab, Fab′, F(ab′)₂, or other antigen-binding subsequencesof antibodies) which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementarity-determining region (CDR) of the recipient are replacedby residues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies can comprise residues which are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and optimizeantibody performance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details see Jones et al., Nature 321:52(1986): Reichmann et al., Nature 332:323 (1988); and Presta, Curr. Op.Struct. Biol. 2:593 (1992).

As noted above, the present invention provides compositions and methodsfor treating a tissue disorder associated with a response-to-injuryprocess or proliferating cells in a mammal. More specifically, basedupon the pathways and progression of disease described herein, it is nowunderstood that many diseases are related in the sense that transitionmolecules are involved in the initiation and progression of disease.Provided in more detail below are: (A) assays for detecting moleculessuitable for use within the present invention; (B) suitable candidatemolecules for use within the present invention (whether for assaying, orfor therapeutic purpose); (C) antibodies (for either assaying, or fortherapeutic purpose); (D) expression systems for producing and ordelivering therapeutic quantities of a desired polypeptide; (E) methodsof treating a wide variety of diseases; and (F) the preparation ofpharmaceutical compositions, including vaccines.

A. Assays for Detecting Therapeutic Molecules

The present invention provides a number of assays suitable for detectingtherapeutic molecules, which are briefly described herein, as well as inmore detail below in the examples.

For example, within one aspect of the invention methods of identifying apeptide or polypeptide composition for treating a tissue disorderassociated with a response-to-injury process, or, the proliferation ofcells in a mammal is provided, comprising the general steps of: (a)selecting a sequence from a database screened for sequences comprising apeptide of the sequence BX7B (SEQ ID NO:28) wherein B is a basic aminoacid, and X7 is a sequence of about seven residues is selected from anyamino acid other than an acidic amino acid, wherein the peptide forms analpha helix and each occurrence of B is oriented on the same side of thealpha helix. (b) preparing a composition comprised of the selectedsequence: and (c) testing the prepared composition for the ability toinhibit podosome formation. Within certain embodiments, the peptide in(a) does not consist of the sequences BBXXBBBXXBB, KQKIKHVVKLK,KLKSQLVKRK, RYPISRPRKR, KNGRYSISR, RDGTRYVQKGEYR, RRRCGQKKK, RGTRSGSTR,RRRKKIQGRSKR, RKSYGKYQGR, KVGKSPPVR, KTFGKMKPR, RIKWSRVSK, KRTMRPTRR,KVGKSPPVR, or HREARSGKYK (SEQ ID Nos. 29-44 respectively).

In a related aspect, the invention provides methods of identifying apeptide or polypeptide composition for treating a tissue disorderassociated with a response-to-injury process or proliferating cells in amammal comprising the steps of: (a) forming an expression librarycomprised of cloned sequences expressed by a cell during a transitionstage response; (b) screening the expression library for sequencesencoding a peptide or polypeptide that binds a hyaladherin that isstimulated in cells during the transition stage; (c) preparing a peptideor polypeptide encoded by the hyaladherin binding sequences; and (d)testing the peptide or polypeptide for the ability to affect at leastone activity associated with transition stage cells wherein the activityis selected from the group consisting of: increased erk kinase signalactivation, podosome formation, metalloproteinase expression, flux ofintracellular or extracellular HA or hyaladherins, expression of ahyaladherin, and inability to form focal adhesions. In one embodiment ofthis method, the expression vector is a two hybrid phage display system,the hyaladherin is RHAMM and the testing is for the ability to inhibitpodosome formation and inhibition of erk kinase signaling activation. Ina related embodiment of these methods, the library is a library ofnucleic acid molecules, or organic molecules, and the library is testedin order to determine its ability to affect one of the activities setforth in (d) above. If the test is positive, the library may bedeconvoluted, and screened until a single molecule is identified.

In still another aspect, the invention provides methods for detectinghyalauronic acid in a sample comprising the steps of: (a) incubating thesample with a hyalauronic acid binding peptide comprising a sequenceselected from the group consisting of SEQ. ID NO: 1-10; and (b)detecting an amount of a complex formed between hyalauronic acid and thehyalauronic acid binding peptide. In one embodiment, the detectingemploys an antibody that specifically binds to the hyalauronic acidbinding peptide.

In a related aspect, the invention provides methods of detecting amolecule that binds to a RHAMM polypeptide in a sample comprising thesteps of (a) incubating the sample with the RHAMM polypeptide and with aRHAMM-binding polypeptide comprised of SEQ. ID NO: 21; and (b) detectingan amount of a complex formed between the sample and the RHAMMpolypeptide by scoring for reduced binding between the RHAMM polypeptideand the RHAMM-binding polypeptide. In one embodiment, this methodincludes detecting which employs an antibody that specifically binds tothe RHAMM-binding polypeptide.

These as well as other methods are described in more detail below in theexamples.

B. Candidate Molecules

Utilizing the assays provided herein, a wide variety of molecules may beassayed for their ability to treat or prevent a tissue disorderassociated with a response-to-injury process or proliferating cells.Representative examples which are discussed in more detail below includeorganic molecules, proteins or peptides, and nucleic acid molecules.

1. Organic Molecules

Numerous organic molecules may be assayed for their ability to treat orprevent a tissue disorder associated with a response-to-injury processor proliferating cells.

For example, within one embodiment of the invention suitable organicmolecules may be selected from either from a chemical library, whereinchemicals are assayed individually, or from combinatorial chemicallibraries where multiple compounds are assayed at once, thendeconvoluted to determine and isolate the most active compounds.

Representative examples of such combinatorial chemical libraries includethose described by Agrafiotis et al., “System and method ofautomatically generating chemical compounds with desired properties.”U.S. Pat. No. 5,463,564; Armstrong, R. W. “Synthesis of combinatorialarrays of organic compounds through the use of multiple componentcombinatorial array syntheses.” WO 95/02566; Baldwin, J. J. et al.,“Sulfonamide derivatives and their use.” WO 95/24186; Baldwin, J. J. etal. “Combinatorial dihydrobenzopyran library,” WO 95/30642; Brenner, S.“New kit for preparing combinatorial libraries.” WO 95/16918; Chenera,B. et al. “Preparation of library of resin-bound aromatic carbocycliccompounds.” WO 95/16712; Ellman. J. A., “Solid phase and combinatorialsynthesis of benzodiazepine compounds on a solid support.” U.S. Pat. No.5,288,514. Felder. E. et al. “Novel combinatorial compound libraries.”WO 95/16209; Lerner, R. et al. “Encoded combinatorial chemicallibraries.” WO 93/20242; Pavia, M. R. et al. “A method for preparing andselecting pharmaceutically useful non-peptide compounds from astructurally diverse universal library.” WO 95/04277; Summerton, J. E.and D. D. Weller. “Morpholino-subunit combinatorial library and method.”U.S. Pat. No. 5,506,337. Holmes, C. “Methods for the Solid PhaseSynthesis of Thiazolidinones, Metathiazanones, and Derivatives thereof,”WO 96/00148. Phillips, G. B. and G. P. Wei, “Solid-phase Synthesis ofBenzimidazoles,” Tet. Letters 37:4887-90, 1996; Ruhland, B. et al.,“Solid-supported Combinatorial Synthesis of Structurally Diverseβ-Lactams,” J. Amer. Chem. Soc. 111:253-4, 1996; Look, G. C. et al.,“The Identification of Cyclooxygenase-1 Inhibitors from 4-ThiazolidinoneCombinatorial Libraries,” Bioorg and Med. Chem. Letters 6:707-12, 1996.

2. Proteins and Peptides

A wide range of proteins and peptides may likewise be assayed for theirability to treat or prevent a tissue disorder associated with aresponse-to-injury process or proliferating cells.

a. Combinatorial Peptide Libraries

Suitable peptide molecules may be obtained through the screening, ofcombinatorial peptide libraries. Such libraries may either be preparedby one of skill in the art (see e.g., U.S. Pat. Nos. 4,528,266 and4,359,535, and Patent Cooperation Treaty Publication Nos. WO 92/15679,WO 92/15677, WO 90/07862, WO 90/0'809, or purchased from commerciallyavailable sources (e.g., New England Biolabs Ph.D.™ Phage DisplayPeptide Library Kit).

b. Peptide Mimetics

Numerous peptide mimetics may also be utilized within the presentinvention, including for example peptides such as: (H4-5)B3; SEQ. ID.NO: 1 (H4-5)BXBBXB: SEQ. ID. NO: 2 (H4-5)BXBXBBB; SEQ. ID. NO: 3(H4-5)BXBBB; SEQ. ID. NO: 4 and (H4-5)BXBB SEQ. ID. NO: 5

where B is either lysine (K) or arginine (R and X is a hydrophobic orneutral amino acid (i.e., L,V,Q,S) and H represents a series of aminoacids such that an alpha helix is formed, as determined by NN-predictEMBL protein analysis. This need not be an amphipathic or coiled coilhelix but such would also be suitable. Specific examples of sequencesfitting these motifs that have been analyzed for effectiveness onpodosome include the following: MMTVLKR; SEQ. ID. NO: 6 MMTVLKVKRLR;SEQ. ID. NO: 7 MMTVLKVKVKRK; SEQ. ID. NO: 8 MMTVLKVRKR: SEQ. ID. NO: 9and MMTVLKVRK. SEQ. ID. NO: 10

In addition, the following RHAMM sequences are more highly exposed oncell surfaces and more effective at blocking podosomes, cell motilityand cell invasion. These are: KLQATQKPLTESK. SEQ ID NO: 11 andVSIEKEKIDEKS. SEQ ID NO: 12

Other peptides may likewise be developed based upon the TAM domain(“Transient Activator of Map kinases”). This sequence is VS(I/L)EKE. SEQID NO: 13

Since this sequence is included in those used to prepare polyclonalantibodies against RHAMM, and because such antibodies blocks cellmotility and activation of erk by growth factors. TAM domains have beenidentified as key sites of protein-protein interaction that are requiredfor controlling map kinase pathways. This in turn regulates theactivation of the cell to migrate, proliferate and remodel extracellularmatrix. Reagents to this sequence will be useful in therapeutictreatment of the diseases described above.

Antibodies, peptide mimics or antisense technology of these motifs canbe used to disrupt the transient phenotype and for treatment of diseaseas described in more detail below.

C. Antibodies

Antibodies, as described in more detail below, may likewise be assayedfor their ability to treat or prevent a tissue disorder associated witha response-to-injury process or proliferating cells.

d. Production of Proteins

Although various genes (or portions thereof) have been provided herein,it should be understood that within the context of the presentinvention, reference to one or more of these genes includes derivativesof the genes that are substantially similar to the genes (and, whereappropriate, the proteins (including peptides and polypeptides) that areencoded by the genes and their derivatives). As used herein, anucleotide sequence is deemed to be “substantially similar” if: (a) thenucleotide sequence is derived from the coding region of theabove-described genes and includes, for example, portions of thesequence or allelic variations of the sequences discussed above. (b) thenucleotide sequence is capable of hybridization to nucleotide sequencesof the present invention under moderate, high or very high stringency(see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.,Cold Spring Harbor Laboratory Press. NY. 1989): or (c) the DNA sequencesare degenerate as a result of the genetic code to the DNA sequencesdefined in (a) or (b). Further, the nucleic acid molecule disclosedherein includes both complementary and non-complementary sequences,provided the sequences otherwise meet the criteria set forth herein.Within the context of the present invention, high stringency meansstandard hybridization conditions (e.g. 5×SSPE, 0.5% SDS at 65° C. orthe equivalent).

The structure of the proteins encoded by the nucleic acid moleculesdescribed herein may be predicted from the primary translation productsusing the hydrophobicity plot function of, for example. P/C Gene orIntelligenetics Suite (Intelligenetics, Mountain View. Calif.), oraccording to the methods described by Kyte and Doolittle (J. Mol. Biol.157:105-132, 1982).

Proteins of the present invention may be prepared in the form of acidicor basic salts, or in neutral form. In addition, individual amino acidresidues may be modified by oxidation or reduction. Furthermore, varioussubstitutions, deletions, or additions may be made to the amino acid ornucleic acid sequences, the net effect of which is to retain or furtherenhance or decrease the biological activity of the mutant or wild-typeprotein. Moreover, due to degeneracy in the genetic code, for example,there may be considerable variation in nucleotide sequences encoding thesame amino acid sequence.

Other derivatives of the proteins disclosed herein include conjugates ofthe proteins along with other proteins or polypeptides. This may beaccomplished, for example, by the synthesis of N-terminal or C-terminalfusion proteins which may be added to facilitate purification oridentification of proteins (see U.S. Pat. No. 4,851,341, see also, Hoppet al. Bio/Technology, 6:1204, 1988.) Alternatively, fusion proteinssuch as Flag/desired protein binding protein be constructed in order toassist in the identification, expression, and analysis of the protein.

Proteins of the present invention may be constructed using a widevariety of techniques described herein. Further, mutations may beintroduced at particular loci by synthesizing oligonucleotidescontaining a mutant sequence, flanked by restriction sites enablingligation to fragments of the native sequence. Following ligation, theresulting reconstructed sequence encodes a derivative having the desiredamino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific (or segmentspecific) mutagenesis procedures may be employed to provide an alteredgene having particular codons altered according to the substitution,deletion, or insertion required. Exemplary methods of making thealterations set forth above are disclosed by Walder et al. (Gene 42:133,1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods,Plenum Press, 1981); and Sambrook et al. (supra). Deletion or truncationderivatives of proteins (e.g. a soluble extracellular portion) may alsobe constructed by utilizing convenient restriction endonuclease sitesadjacent to the desired deletion. Subsequent to restriction, overhangsmay be filled in, and the DNA religated. Exemplary methods of making thealterations set forth above are disclosed by Sambrook et al. (MolecularCloning. A Laboratory Manual, 2d Ed., Cold Spring Harbor LaboratoryPress, 1989).

Mutations which are made in the nucleic acid molecules of the presentinvention preferably preserve the reading frame of the coding sequences.Furthermore, the mutations will preferably not create complementaryregions that could hybridize to produce secondary mRNA structures, suchas loops or hairpins, that would adversely affect translation of themRNA. Although a mutation site may be predetermined, it is not necessarythat the nature of the mutation per se be predetermined. For example, inorder to select for optimum characteristics of mutants at a given site,random mutagenesis may be conducted at the target codon and theexpressed mutants screened for indicative biological activity.Alternatively, mutations may be introduced at particular loci bysynthesizing oligonucleotides containing a mutant sequence, flanked byrestriction sites enabling ligation to fragments of the native sequence.Following ligation, the resulting reconstructed sequence encodes aderivative having the desired amino acid insertion, substitution, ordeletion.

Nucleic acid molecules which encode proteins of the present inventionmay also be constructed utilizing techniques of PCR mutagenesis,chemical mutagenesis (Drinkwater and Klinedinst, PNAS 83:3402-3406,1986), by forced nucleotide misincorporation (e.g., Liao and Wise Gene88:107-111, 1990), or by use of randomly mutagenized oligonucleotides(Horwitz et al., Genome 3:112-117, 1989).

The present invention also provides for the manipulation and expressionof the above described genes by culturing host cells containing a vectorcapable of expressing the above-described genes. Such vectors or vectorconstructs include either synthetic or cDNA-derived nucleic acidmolecules encoding the desired protein, which are operably linked tosuitable transcriptional or translational regulatory elements. Suitableregulatory elements may be derived from a variety of sources, includingbacterial, fungal, viral, mammalian, insect, or plant genes. Methods forexpressing genes of interest are describe in more detail above.

Proteins can be isolated by, among other methods, culturing suitablehost and vector systems to produce the recombinant translation productsof the present invention. Supernatants from such cell lines, or proteininclusions or whole cells where the protein is not excreted into thesupernatant, can then be treated by a variety of purification proceduresin order to isolate the desired proteins. For example, the supernatantmay be first concentrated using commercially available proteinconcentration filters, such as an Amicon or Millipore Pelliconultrafiltration unit. Following concentration, the concentrate may beapplied to a suitable purification matrix such as, for example, ananti-protein antibody bound to a suitable support. Alternatively, anionor cation exchange resins may be employed in order to purify theprotein. As a further alternative, one or more reverse-phase highperformance liquid chromatography (RP-HPLC) steps may be employed tofurther purify the protein. Other methods of isolating the proteins ofthe present invention are well known in the skill of the art.

A protein is deemed to be “isolated” within the context of the presentinvention if no other (undesired) protein is detected pursuant toSDS-PAGE analysis followed by Coomassie blue staining. Within otherembodiments, the desired protein can be isolated such that no other(undesired) protein is detected pursuant to SDS-PAGE analysis followedby silver staining.

3. Nucleic Acid Molecules

Within other aspects of the invention, nucleic acid molecules can beassayed for their ability to treat or prevent a tissue disorderassociated with a response-to-injury process or proliferating cells. Forexample, within one embodiment antisense oligonucleotide molecules areprovided which specifically inhibit expression of nucleic acid sequenceswhich are associated with a response-to injury process or theproliferation of cells (see generally, Hirashima et al. in MolecularBiology of RNA. Nell, Perspectives (M. Inouye and B. S. Dudock, eds.,1987 Academic Press, San Diego, p. 401); Oligonucleotides: AntisenseInhibitors of Gene Expression (J. S. Cohen, ed., 1989 MacMillan Press,London); Stein and Cheng, Science 261:1004-1012, 1993: WO 95/10607; U.S.Pat. No. 5,359,051; WO 92/06693; and EP-A2-612844). Briefly, suchmolecules are constructed such that they are complementary to, and ableto form Watson-Crick base pairs with, a region of a transcribed mRNAsequence. The resultant double-stranded nucleic acid interferes withsubsequent processing of the mRNA, thereby preventing protein synthesis.

Within other aspects of the invention, ribozymes are provided which arecapable of inhibiting the expression of sequences which are associatedwith, or which encode proteins or polypeptides that are associated withthe disorders described herein. As used herein. “ribozymes” are intendedto include RNA molecules that contain anti-sense sequences for specificrecognition, and an RNA-cleaving enzymatic activity. The catalyticstrand cleaves a specific site in a target RNA at greater thanstoichiometric concentration. A wide variety of ribozymes may beutilized within the context of the present invention, including forexample, the hammerhead ribozyme (for example, as described by Forsterand Symons, Cell 48: 211-220, 1987: Haseloff and Gerlach, Nature328:596-600, 1988; Walbot and Bruening, Nature 334:196, 1988; Haseloffand Gerlach, Nature 334:585, 1988); the hairpin ribozyme (for example,as described by Haseloff et al., U. S. Pat. No. 5,254,678, issued Oct.19, 1993 and Hempel et al. European Patent Publication No. 0 360 257,published Mar. 26, 1990); and Tetrahymena ribosomal RNA-based ribozymes(see Cech et al. U.S. Pat. No. 4,987,071). Ribozymes of the presentinvention typically consist of RNA, but may also be composed of DNA,nucleic acid analogs (e.g. phosphorothioates), or chimerics thereof (eg. DNA/RNA/RNA).

4. Labels

The gene product or any of the candidate molecules described above andbelow, may be labeled with a variety of compounds, including forexample, fluorescent molecules, toxins, and radionuclides.Representative examples of fluorescent molecules include fluorescein,Phycobili proteins, such as phycoerythrin, rhodamine. Texas red andluciferase. Representative examples of toxins include ricin, abrindiphtheria toxin, cholera toxin, gelonin, pokeweed antiviral protein,tritin, Shigella toxin, and Pseudomonas exotoxin A. Representativeexamples of radionuclides include Cu-64, Ga-67. Ga-68, Zr-89, Ru-97,Tc-99m, Rh-105, Pd-109, In-111, I-123, I-125, I-131, Re-186, Re-188,Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. In addition, theantibodies described herein may also be labeled or conjugated to onepartner of a ligand binding pair. Representative examples includeavidin-biotin, and riboflavin-riboflavin binding protein.

Methods for conjugating or labeling the molecules described herein withthe representative labels set forth above may be readily accomplished byone of ordinary skill in the art (see Trichothecene Antibody Conjugate,U.S. Pat. No. 4,744,981: Antibody Conjugate, U.S. Pat. No. 5,106,951:Fluorogenic Materials and Labeling (Techniques, U.S. Pat. No. 4,018,884:Metal Radionuclide Labeled Proteins for Diagnosis and Therapy. U.S. Pat.No. 4,897,255: and Metal Radionuclide Chelating Compounds for ImprovedChelation Kinetics. U.S. Pat. No. 4,988,496: see also Inman, Methods InEnzymology, Vol. 34, Affinity Techniques, Enzyme Purification: Part B.Jakoby and Wilchek, (eds.). Academic Press, New York, p. 30, 1974: seealso Wilchek and Bayer, “The Avidin-Biotin Complex in BioanalyticalApplications,” Anal. Biochem. 171:1-32.1988).

C. Antibodies

Antibodies to the polypeptides, fragments, or peptides described hereinmay readily be prepared by one of skill in the art given the disclosureprovided herein. Within the context of the present invention, the term“antibody” should be understood to include monoclonal antibodies,polyclonal antibodies, anti-idiotypic antibodies, antibody fragments(e.g. Fab, and F(ab′)₂. F_(V) variable regions, or complementarydetermining regions), whether obtained from animals or humans, generatedutilizing hybridoma technology, or recombinantly produced. Antibodiesare generally accepted as specific against an antigen if they bind witha K_(d) of at least 10⁻⁷ M (moles/liter), and more preferably, at least10⁻⁸ M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, 10⁻¹² M, 10⁻¹³ M, 10⁻¹³ M, or, 10⁻¹⁴M. The affinity of a monoclonal antibody or binding partner can bereadily determined by one of ordinary skill in the art (see Scatchard,Ann. N.Y. Acad. Sci. 51:660-672, 1949). Antibodies of the presentinvention should also bind to the desired domain or peptide with thespecificity noted above, and not against randomized peptides.

Briefly, a polyclonal antibody preparation may be readily generated in avariety of warm-blooded animals such as rabbits, mice, or rats.Typically, an animal is immunized with a desired antigen or peptidethereof, which may be conjugated to a carrier protein, such as keyholelimpet hemocyanin. Routes of administration include intraperitoneal,intramuscular, intraocular, or subcutaneous injections, usually in anadjuvant (e.g. Freund's complete or incomplete adjuvant). Particularlypreferred polyclonal antisera demonstrate binding in an assay that is atleast three times greater than background.

Monoclonal antibodies may also be readily generated from hybridoma celllines using conventional techniques (see U.S. Pat. Nos. RE 32,011,4,902,614, 4,543,439, and 4,411,993; see also Antibodies: A LaboratoryManual, Harlow and Lane (eds.), Cold Spring Harbor Laboratory Press,1988). Briefly, within one embodiment, a subject animal such as a rat ormouse is injected with an antigen of interest or a portion thereof. Theprotein may be administered as an emulsion in an adjuvant such asFreund's complete or incomplete adjuvant in order to increase the immuneresponse. Between one and three weeks after the initial immunization theanimal is generally boosted and may tested for reactivity to the proteinutilizing well-known assays. The spleen and/or lymph nodes are harvestedand immortalized. Various immortalization techniques, such as mediatedby Epstein-Barr virus or fusion to produce a hybridoma may be used. In apreferred embodiment, immortalization occurs by fusion with a suitablemyeloma cell line (e.g., NS-1 (ATCC No. TIB 18), and P3X63-Ag 8.653(ATCC No. CRL 1580) to create a hybridoma that secretes monoclonalantibody. The preferred fusion partners do not express endogenousantibody genes. Following fusion, the cells are cultured in mediumcontaining a reagent that selectively allows for the growth of fusedspleen and myeloma cells such as HAT (hypoxanthine, aminopterin, andthymidine) and are subsequently screened for the presence of antibodiesthat are reactive against the desired anti-en of interest. A widevariety of assays may be utilized, including for example countercurrentimmuno-electrophoresis, radioimmunoassays, radioimmunoprecipitations,enzyme-linked immunosorbent assays (ELISA), dot blot assays, westernblots, immunoprecipitation, inhibition or competition assays, andsandwich assays (see U.S. Pat. Nos. 4,376,110 and 4,486,530; see alsoAntibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988).

Other techniques may also be utilized to construct monoclonal antibodies(see Huse et al., Science 246:1275-1281, 1989; Sastry et al., Proc.Natl. Acad. Sci. USA 86:5728-5732, 1989; Alting-Mees et al., Strategiesin Molecular Biology 3:1-9, 1990: describing recombinant techniques).Briefly. RNA is isolated from a B cell population and utilized to createheavy and light chain immunoglobulin cDNA expression libraries insuitable vectors, such as λImmunoZap(H) and λImmunoZap(L). These vectorsmay be screened individually or co-expressed to form Fab fragments orantibodies (see Huse et al., supra; Sastry et al., supra). Positiveplaques may subsequently be converted to a non-lytic plasmid that allowshigh level expression of monoclonal antibody fragments from E coli.

Similarly, portions or fragments, such as Fab and Fv fragments, ofantibodies may also be constructed utilizing conventional enzymaticdigestion or recombinant DNA techniques to yield isolated variableregions of an antibody. Within one embodiment, the genes which encodethe variable region from a hybridoma producing a monoclonal antibody ofinterest are amplified using nucleotide primers for the variable region.These primers may be synthesized by one of ordinary skill in the art, ormay be purchased from commercially available sources (e.g. Stratacyte,La Jolla, Calif.) Amplification products are inserted into vectors suchas ImmunoZAP™ H or ImmunoZAP™ L (Stratacyte), which are then introducedinto E coli, yeast, or mammalian-based systems for expression. Utilizingthese techniques, large amounts of a single-chain protein containing afusion of the V_(H) and V_(L) domains may be produced (see Bird et al.,Science 242:423-426, 1988). In addition, techniques may be utilized tochange a “murine” antibody to a “human” antibody, without altering thebinding specificity of the antibody. Examples of humanized antibodiesinclude chimeric or CDR-grafted antibodies (U.S. Pat. Nos. 4,816,567 and5.225,539), antibodies produced in genetically-altered mice (see PCTApplication No. 93/12227).

One of ordinary skill in the art will appreciate that a variety ofalternative techniques for generating antibodies exist. In this regard,the following U.S. patents teach a variety of these methodologies andare thus incorporated herein by reference: U.S. Pat. Nos. 5,840,479;5,770,380; 5,204,244; 5,482,856; 5,849,288; 5,780,225; 5,780,225;5,395,750; 5,225,539; 5,110,833; 5,693,762; 5,693,761; 5,693,76;5,698,435; and 5,328,834.

Once suitable antibodies have been obtained, they may be isolated orpurified by many techniques well known to those of ordinary skill in thean (see Antibodies: A Laboratory Manual, Harlow and Lane (eds.). ColdSpring Harbor Laboratory Press, 1988). Suitable techniques includepeptide or protein affinity columns, HPLC (e.g., reversed phase, sizeexclusion, ion-exchange), purification on protein A or protein Gcolumns, or any combination of these techniques.

D. Expression Systems

1. Vectors, Host Cells and Means of Expressing and Producing Protein

Proteins or polypeptides of the present invention may be readilyexpressed in a variety of host cells or organisms. For proteinproduction and purification, proteins are preferably secreted andproduced in bacteria, such as E. coli, for which many expression vectorshave been developed and are available. Other suitable host organismsinclude other bacterial species (e.g., Bacillus, and eukaryotes, such asyeast (e.g., Saccharomyces cerevisiae), mammalian cells (e.g., CHO andCOS-7), plant cells and insect cells (e.g., Sf9). Vectors for thesehosts are well known.

Briefly, within one embodiment a DNA sequence encodings a desiredprotein or polypeptide is introduced into an expression vectorappropriate for the host. The sequence is derived from an existing cloneor synthesized. A preferred means of synthesis is amplification of thegene from cDNA, genomic DNA, or a recombinant clone using a set ofprimers that flank the coding region or the desired portion of theprotein. Restriction sites are typically incorporated into the primersequences and are chosen with regard to the cloning site of the vector.If necessary, translational initiation and termination codons can beengineered into the primer sequences. The desired sequence can becodon-optimized for expression in a particular host. For example, asecreted form of a desired protein that is expressed in a fungal host,such as yeast, can be altered in nucleotide sequence to use codonspreferred in yeast. Codon-optimization may be accomplished by methodssuch as splice overlap extension, site-directed mutagenesis, automatedsynthesis, and the like.

At minimum, the vector must contain a promoter sequence. Otherregulatory sequences however may also be included. Such sequencesinclude, a transcription termination signal sequence, secretion signalsequence, origin of replication, selectable marker, and the like. Theregulatory sequences are operationally associated with one another toallow transcription or translation.

2. Expression in Bacteria

The plasmids used herein for expression of a desired protein orpolypeptide include a promoter designed for expression of the proteinsin a bacterial host. Suitable promoters are widely available and arewell known in the art. Inducible or constitutive promoters arepreferred. Such promoters for expression in bacteria include promotersfrom the T7 phage and other phages, such as T3. T5, and SP6, and thetrp, lpp, and lac operons. Hybrid promoters (see, U.S. Pat. No.4,551,433), such as tac and tre, may also be used. Promoters forexpression in eukaryotic cells include the P10 or polyhedron genepromoter of baculovirus/insect cell expression systems (see, e.g., U.S.Pat. Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784).MMTV LTR, RSV LTR, SV40, metallothionein promoter (see, e.g. U.S. Pat.No. 4,870,009) and other inducible promoters. For expression of theproteins, a promoter is inserted in operative linkage with the codingregion of the desired protein or polypeptide.

The promoter controlling transcription of the desired protein may becontrolled by a repressor. In some systems, the promoter can bederepressed by altering the physiological conditions of the cell, forexample, by the addition of a molecule that competitively binds therepressor, or by altering the temperature of the growth media. Preferredrepressor proteins include, but are not limited to the E. coli ladrepressor responsive to IPTG induction, the temperature sensitive λc1857repressor, and the like. The E. coli lacI repressor is preferred.

In other preferred embodiments, the vector also includes a transcriptionterminator sequence. A “transcription terminator region” has either asequence that provides a signal that terminates transcription by thepolymerase that recognizes the selected promoter and/or a signalsequence for polyadenylation.

Preferably, the vector is capable of replication in bacterial cells.Thus, the vector preferably contains a bacterial origin of replication.Preferred bacterial origins of replication include the f1-ori and col E1origins of replication, especially the ori derived from pUC plasmids.

The plasmids also preferably include at least one selectable marker thatis functional in the host. A selectable marker gene includes any genethat confers a phenotype on the host that allows transformed cells to beidentified and selectively grown. Suitable selectable marker genes forbacterial hosts include the ampicillin resistance gene (Amp^(r)),tetracycline resistance gene (Tc^(r)) and the kanamycin resistance gene(Kan^(r)). Suitable markers for eukaryotes usually require acomplementary deficiency in the host (e.g. thymidine kinase (tk) in tk−hosts). However, drug markers are also available (e.g., G418 resistanceand hygromycin resistance).

The sequence of nucleotides encoding the desired protein or polypeptidemay also include a classical secretion signal, whereby the resultingpeptide is a precursor protein processed and secreted. The resultingprocessed protein may be recovered from the periplasmic space or thefermentation medium. Secretion signals suitable for use are widelyavailable and are well known in the art (von Heijne, J. Mol. Biol.184:99-105. 1985). Prokaryotic and eukaryotic secretion signals that arefunctional in E. coli (or other host) may be employed. The presentlypreferred secretion signals include, but are not limited to pelB, matα,extension and glycine-rich protein.

One skilled in the art appreciates that there are a wide variety ofsuitable vectors for expression in bacterial cells and which are readilyobtainable. Vectors such as the pET series (Novagen, Madison, Wis.) andthe tac and trc series (Pharmacia. Uppsala. Sweden) are suitable forexpression of a wide variety of proteins. A suitable plasmid isampicillin resistant, has colEI origin of replication, lacI^(q) gene, alac/trp hybrid promoter in front of the lac Shine-Dalgarno sequence, ahexa-his coding sequence that joins to the 3′ end of the inserted gene,and an rrnB terminator sequence.

The choice of a bacterial host for the expression of the desired proteinor polypeptide is dictated in part by the vector. Commercially availablevectors are paired with suitable hosts. The vector is introduced inbacterial cells by standard methodology. Typically, bacterial cells aretreated to allow uptake of DNA (for protocols, see generally, Ausubel etal., supra; Sambrook et al., supra). Alternatively, the vector may beintroduced by electroporation, phage infection, or another suitablemethod.

3. Expression in Other Organisms

A variety of other organisms are suitable for use in the presentinvention. For example, various fungi, including yeasts, molds, andmushrooms, insects, especially vectors for diseases and pathogens, andother animals, such as cows, mice, goats, birds, aquatic animals (e.g.,shrimp, turtles, fish, lobster and other crustaceans), amphibians andreptiles and the like, may be transformed with a desired transgene.

The principles that guide vector construction for bacteria and plants,as discussed above, are applicable to vectors for these organisms. Ingeneral, vectors are well known and readily available. Briefly, thevector should have at least a promoter functional in the host inoperative linkage with the desired protein or polypeptide. Usually, thevector will also have one or more selectable markers, an origin ofreplication, a polyadenylation signal and transcription terminator.

The sequence of nucleotides encoding the desired protein or polypeptidemay also include a classical secretion signal, whereby the resultingpeptide is a precursor protein processed and secreted. Suitablesecretion signals may be obtained from a variety of genes, such asmat-alpha or invertase genes.

4. Transgenic Animals

Within related aspects of the present invention, proteins of the presentinvention may be expressed in a transgenic animal whose germ cells andsomatic cells contain a gene which encodes the desired protein and whichis operably linked to a promoter effective for the expression of thegene. Alternatively, in a similar manner transgenic animals may beprepared that lack the desired gene (e.g., “knockout” mice). Suchtransgenics may be prepared in a variety non-human animals, includingmice, rats, rabbits, sheep, dogs, goats and pigs (see Hammer et al.,Nature 315:680-683, 1985, Palmiter et al, Science 222:809-814, 1983.Brinster et al. Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985, Palmiterand Brinster, Cell 41:343-345, 1985, PCT Publication No. WO 99/01164,and U.S. Pat. Nos. 5,175,383, 5,087,571, 4,736,866, 5,387,742.5,347,075, 5,221,778, 5,162,215; 5,545,808; 5,741,957; 4,873,191;5,780,009; 4,736,866; 5,567,607; 5,633,076 and 5,175,384). Briefly, anexpression vector, including a nucleic acid molecule to be expressedtogether with appropriately positioned expression control sequences, isintroduced into pronuclei of fertilized eggs, for example, bymicroinjection. Integration of the injected DNA is detected by blotanalysis of DNA from tissue samples. It is preferred that the introducedDNA be incorporated into the germ line of the animal so that it ispassed on to the animal's progeny. Tissue-specific expression may beachieved through the use of a tissue-specific promoter, or through theuse of an inducible promoter, such as the metallothionein gene promoter(Palmiter et al., 1983, ibid), which allows regulated expression of thetransgene.

E. Gene Delivery Vectors

A wide variety of gene delivery vectors may be utilized to deliverand/or express a desired gene of interest in host cells. For example,within one aspect of the present invention, retroviral gene deliveryvehicles may be utilized. Briefly, retroviral gene delivery vehicles ofthe present invention may be readily constructed from a wide variety ofretroviruses, including for example. B. C, and D type retroviruses aswell as spumaviruses and lentiviruses (see RNA Tumor Viruses, SecondEdition. Cold Spring Harbor Laboratory, 1985). Such retroviruses may bereadily obtained from depositories or collections such as the AmericanType Culture Collection (“ATCC”; Rockville, Md.), or isolated from knownsources using commonly available techniques. Representative examples ofretroviral gene delivery vectors are described in more detail in EP0,415,731: PCT Publication Nos. WO 90/07936: WO 91/0285. WO 93/1230: WO93/0218. WO 9403622; WO 9325698; WO 9325234, and U.S. Pat. Nos.5,219,740, 5,716.613, 5,851,529, 5,591,624, 5,716,826, 5,716,832, and5,817,491.

Other suitable gene delivery vectors can be generated from alphaviruses(see e.g., U.S. Pat. Nos. 5,091,309 and 5,217,879, 5,843,723, and5,789,245), recombinant adenoviral vectors (see e.g., U.S. Pat. No.5,872,005), and numerous other viruses such as pox viruses, such ascanary pox virus or vaccinia virus (Fisher-Hoch et al. PNAS 86:317-321,1989; Flexner et al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner etal., Vaccine 8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and5,017,487: WO 89/01973); SV40 (Mulligan et al., Nature 277:108-114,1979); influenza virus (Luytjes et al., Cell 59:1107-1113, 1989;McMicheal et al. N. Eng. J. Med. 309:13-17, 1983; and Yap et al., Nature273:238-239, 1978); herpes (Kit. Adv. Exp. Med. Biol. 21.5:2-19-236,1989; U.S. Pat. No. 5,288,641); HIV (Poznansky, J. Virol. 65:532-536,1991): measles (EP 0 440,219); Semliki Forest Virus, and coronavirus, aswell as other viral systems (e.g. EP 0,440,219; WO 92/06693; U.S. Pat.No. 5,166,057).

In addition to the above viral-based vectors, numerous non-viral genedelivery vehicles may likewise be utilized within the context of thepresent invention. Representative examples of such gene deliveryvehicles include direct delivery of nucleic acid expression vectors ornaked DNA alone (see e.g. U.S. Pat. Nos. 5,814,482 and 5,580,859),polycation condensed DNA linked or unlinked to killed adenovirus (Curielet al., Hum. Gene Ther. 3:147-154, 1992), DNA ligand linked to a ligand(Wu et al., J. of Biol. Chem. 264:16985-16987, 1989), and nucleic acidcontaining liposomes (e.g., WO 95/24929 and WO 95/12387).

F. Compounds

As noted above, a wide variety of compounds may be utilized within thisregard, including for example (a) a polypeptide comprising the aminoacid sequence BX7B (SEQ ID NO:28) which binds HA; (b) an anti-TAMantibody: (c) a polypeptide fragment which encodes a D1. D2. D3. D4, or.D5 domain of RHAMM; and (d) a gene delivery vector which expressesantisense RHAMM, or, delivers and expresses any one of (a), (b), or (c).

Within one embodiment, the polypeptide BX7B (SEQ ID NO:28) comprises apolypeptide wherein B is a basic amino acid and X7 is a sequence ofabout seven residues selected from any amino acid other than an acidicamino acid, wherein the peptide forms an alpha helix and each occurrenceof B is oriented on the same side of the alpha helix, and with theproviso that the peptide does not consist of the sequences BBXXBBBXXBB,KQKIKHVVKLK, KLKSQLVKRK, RYPISRPRKR. KNGRYSISR, RDGTRYVQKGEYR,RRRCGQKKK, RGTRSGSTR, RRRKKIQGRSKR, RKSYGKYQGR, KVGKSPPVR, KTFGKMKPR,RIKWSRVSK, KRTMRPTRR. KVGKSPPVR, or HREARSGKYK (SEQ ID Nos. 29-44respectively.)

In one embodiment, the polypeptide can be (a) a first peptide comprisedof a hyaluronic acid-binding domain; (b) a hyaladherin polypeptide: (c)a second peptide comprised of a domain from a hyaladherin polypeptide:(d) a hyaladherin-binding polypeptide; (e) a third peptide comprised ofa hyaladherin binding domain. Also provided are antibodies which bindsto a peptide or polypeptide of (a)-(d): and/or vectors (e.g., genedelivery vectors described below) that expresses a gene encoding apolypeptide as described above or herein. In a particular embodiment,peptides are provided comprised of a sequence selected from the groupconsisting of SEQ. ID NO: 1-20. In another embodiment, ahyaladherin-binding polypeptide comprised of SEQ. ID NO: 21.

Within particularly preferred embodiments of the invention, the compoundis an antibody. Representative examples of antibodies suitable for usewithin the present invention include antibodies to domain D1 of RHAMMamino acids 1-164 of human RHAMM (including for example: sequencesrecognizing the murine D1 sequence, aa. 97-111-QERGTQDKRIQDME (SEQ IDNO:21); and sequences recognizing human RHAMM, aa 151-164-LKSKFSENGNQKNL(SEQ ID NO:18)); antibodies to domain D2 of RHAMM—the “leucine zipper”domain of human RHAMM from aa 195-222; antibodies which recognize thedomain D3—the TAM domains of RHAMM (aa 219-240 of the human RHAMMsequence, including antibodies which recognize the sequence VSIEKEKIDEK(SEQ ID NO:49)); domain D4 (repeat or “R” domain—aa 442-546 for mouse,and aa 442-4663 for human) and domain D5 (HA binding domain, includingtwo domains: aa 721-730 and aa 742-752 for mouse: aa 635-645 and aa657-666 for human). In other embodiments, antibodies are provided whichbind to a polypeptide comprised of SEQ. ID NO: 11-20.

As utilized herein, reference may be made to the human sequence of RHAMMfor identification of the domains. However, the domains can beidentified and specific antibodies generated for other species, such as,for example, mouse. FIG. 50 (SEQ ID Nos. 47 and 48) provides the aminoacid sequence of human and mouse RHAMM (see PCT publication No. WO97/38098 and Genbank Accession Nos. AAC52049 & Q00547). As utilizedherein, it should be understood that antibodies “bind” to the abovesequence if they do so with a K_(d) of at least 10⁻⁷ M (moles/liter)(see antibodies above).

Also provided are polypeptides comprising a fragment of the RHAMMprotein, of less than 95 or 73 kD molecular weight. Representativefragments of polypeptides contain at least all, or a portion of one ofdomains D1. D2. D3. D4, or D5, as set forth above. Within variousembodiments, the polypeptides are less than 250, 200, 150, 100, 75, 50,or, 25 amino acids in length.

G. Methods of Treatment

As described in more detail below, a wide variety of diseases sharecommon disease processes such as local production of cytokines,degradative enzymes, reactive oxygen species resulting in increased cellmigration and proliferation and eventual tissue destruction and celldeath. These diseases may be readily treated or prevented, byadministration of a composition that alters the activity of transitionmolecules within a cell Transition molecules are comprised ofhyaladherins, hyalauronans or molecules regulated by an amount ofintracellular or extracellular hyaladherins or hyalauronans. Theactivity of hyaladherins and hyalauronans are shown to interact with aregulatory processes associated with a response to injury and/orproliferative/invasive cell types.

In one embodiment, methods are provided comprising the general steps ofadministering to a mammal an effective amount of a composition comprisedof any one of (a) a first peptide comprised of a hyaluronic acid-bindingdomain; (b) a hyaladherin polypeptide; (c) a second peptide comprised ofa domain from a hyaladherin polypeptide; (d) a hyaladherin-bindingpolypeptide; (e) a third peptide comprised of a hyaladherin bindingdomain; (f) an antibody that binds to a peptide or polypeptide of(a)-(e); and/or (g) a vector that expresses a gene encoding any of(a)-(f).

Briefly, cells in a homeostatic environment such as normally occurswithin adult tissues are characterized by a differentiated state thatvaries with the tissue, and a low or contained rate of cellproliferation or motility. This differentiated state has typically beenviewed to occur as a result of specific gene regulation. As described inmore detail below, differentiated cells that are functioning within aphysiological, homeostatic tissue environment are also restricted fromexpressing genes that regulate response-to-injury processes. Theseprocesses are regulated by master switch transcription heterodimerstermed AP-1 as illustrated in FIG. 1. The three map kinase cascadesidentified in mammalian cells so far include erk, jnk and p38 hogpathways. These pathways collectively regulate expression of thetranscription factors c-fos and c-jun. Heterodimerization of these twotranscription factors results in formation of AP-1 which controls theexpression of genes required for cell migration, cell proliferation,extracellular matrix remodeling and production of growth factors andcytokines that are required for amplification and maintenance of theresponse to injury process. It is the deregulated activation of thesepathways that leads to the diseased state.

These transcription factors when activated control the expression of aplethora of molecules required for efficient repair and includeproteases such as collagenases, various extracellular matrix proteinsand molecules that allow the cell to respond to cytokines/growth factorsby proliferating and migrating efficiently. Response-to-injury is awell-defined term referring to the ability of cells to repair and toremodel their extracellular environment to promote and ultimatelyre-establish the differentiated state.

As shown in more detail in the Figures, nomal cells undergo a number ofintermediate changes until it becomes a diseased cell involved inchronic inflammatory diseases, proliferating diseases and degenerativediseases. In FIGS. 1, 2, 3, and 5, we show schematically a model for thekey transition steps involved in the transformation of normal cell todiseased cells which is applicable to all cell types surrounded bymatrix and is most likely involved in all diseases including cancers,inflammatory and degenerative diseases, wound healing and injury relateddiseases, inflammatory implications of host verses graft or devices.Briefly, in the normal tissue, cells are quiescent and responsible fornormal and controlled tissue remodelling. Normal cells express growthfactor receptors (represented by small circles) but these are notgrouped together and the cell is restricted in its ability to respond topro-inflammatory cytokines and growth factors. Rather, the cell remainsin a non-proliferative state and responds to factors that regulate itsstate of differentiation (homeostatic responses). Upon injury, the cellrapidly releases glycosylphosphatidyl inositol linked proteins(co-receptors indicated by triangles) onto the cell surface and releaseshyaluronic acid (represented by X) and other matrix molecules such asfibronectin (represented by ˜) that allow the cell to initiate thebeginnings of an activated state. The presence of the coreceptorspermits growth factors to aggregate slightly enhancing their ability torespond to pro-inflammatory cytokines and growth factors yet at the sametime preventing the full response that is seen in the fully diseasedcell. This regulated ability to respond to growth factors as well as theproduction of molecules such as hyaluronic acid and fibronectin allowsthe formation of podosomes (represented by small triangular extensions)that facilitate the localized release of proteases and other enzymesthat produce fragments of extracellular matrix. These fragments serve torecruit other cells to the site of injury including white cells thatallow enhancement and stabilization of the response to injury.Furthermore, these fragments contribute to the evolution of podosomes tofocal contacts. This intermediary transitional state is termed Stage Cand reagents prepared against the molecules regulating podosomestructure and function are predicted to prevent development of the nextstate. Stare D which is one that allows full responses topro-inflammatory cytokines and growth factors. In Stage D, growth factorand cytokine receptors are aggregated into structures called focalcontacts, which contain all the signaling molecules required foractivation of multiple pathways. In this aggregated state, cells areable to maximally respond to growth factors and cytokines andmaintenance of this state leads to disease.

Cytokines and other pro-inflammatory mediators are not capable ofstimulating the expression of AP-1 dependent genes involved in cellproliferation migration, and tissue destruction. However upon injury; orstress, the cells under go a series of changes which result in thetransformation of normal cells to diseased cells. While not being boundby theory, it is proposed herein that cells that are initiallyresponding to stress, whether due to heat, chemical, free radical,mechanical injury or to mutations of key proteins, react to theseinsults in a standard pattern. The initial stage involves the expressionand secretion of matrix molecules involved in edema and inflammatoryresponses (for example hyaluronic acid (HA), collagen type VIII,osteopontin, tenascin, serglycin, addressin, laminin), as well asexpression of transition molecules on the cell membrane and surface suchas heat shock proteins and HA-binding proteins (Stage B). It is knownthat differentiated cells undergoing transition respond initially toinjury by activating ERK kinase cascades that regulate at the least,activation of heat shock protein transcription factors and potentiallyother transition molecules allowing cells to remain viable as Stage Bcells. These cells are characterized by enhanced production of heatshock proteins that protect the cell from aggregation of key proteins,organelle damage and ultimately apoptosis, as well as by increasedpresence of HA-binding molecules on the cell surface.

Once the cell has entered stage B, the presence of growth factorconcentrations and other molecules at the site of injury will likelydetermine whether the cell now returns to its differentiated state orproceeds to Stage C. The present invention provides the unexpecteddiscovery that closely following the initial responses, cells enter atransitional stage (defined as Stage C) which is characterized by (1)the formation of transient structures called podosomes or invadapodia:(2) disassembled actin cytoskeleton (e.g., a paucity of focal adhesion);and (3) dependence upon hyaluronan related molecules and hyaladherinsfor regulation of signaling cascades; and (4) altered control of growthfactor initiated signaling. As illustrated in the Examples, cells platedonto plastic transiently form podosomes at 12-18 h. but this is reducedby 24 h. Plating of cells onto fibronectin enhances and stabilizespodosome formation.

Fibronectin is also necessary for the formation of focal contacts. Thepresence of podosomes correlates with cell surface RHAMM expression.Such cells importantly exhibit the first stave of release from therestriction of AP-1 activation exhibited by Stage A cells. Podosomesallow the cell to efficiently release proteases at lamellae tips topromote cell invasion into the matrix that will ultimately initiatecontrolled remodelling of its extracellular matrix detected by exposureof the CS-1 sequence in fibronectin (described in more detailhereafter). This event is believed to be required for an ability tomaximally respond to growth factors/cytokines and re-establishment oftissue homeostasis. Podosomes are also ultimately the sites of focaladhesion assembly that ultimately allow a cell to proceed to Stage D. Atthe podosome sites, increased and persistent matrix degradation resultsin increased degradation fragments of matrix molecules such as collagenand CS-1 fibronectin which suppresses the expression and levels of cellsurface transitions molecules. Podosomes require interactions betweenhyaluronan and hyaladherins as well as interactions between hyaladherinsand other proteins for their structural and functional integrity. As thelevels of transitions molecules such as HA-binding molecules (e.g.,RHAMM. CD37) on the cell surface and cytoplasm decrease, there is anincrease in the formation of focal adhesions and local accumulation ofcell surface cytokine receptors, intracellular signaling molecules andcytoskeletal components (Stage D). Focal Adhesions couple integrins togrowth factor/cytokine receptors and allow the cell to enter the nextstage in the injury response which is characterized by heightenedability to respond to pro-inflammatory cytokines and growth factors. Theformation of focal adhesions removes restriction of activation of AP-1dependent genes by cytokines and growth factors and results in increasedcell migration and proliferation, and tissue destruction.

As described in more detail below, the sustained presence of thesecells, termed Stage D are largely responsible for tissue deteriorationfollowing sustained and escalated response to including that ischaracteristic of many inflammatory, degenerative and proliferative typeof diseases including for instance arthritis, multiple sclerosis,psoriasis, inflammatory bowel diseases, restenosis, fibronosis,atherosclerosis, diabetes, osteoarthritis, cancers. Alzheimer's,Parkinson's and wound healing.

These transitional stages (Stages B-C) are necessary for alldifferentiated cells in tissues to activate AP-1 dependent genes andAP-1 dependent disease processes such as cell proliferation, migration,invasion and production of matrix metalloproteinases, therefore,inflammatory, proliferative and degenerative diseases are dynamicprocesses that involve the continual recruitment of differentiatedtissue into the pathway culminating in the stage D cells. The ability ofthe cell to acquire a transitional phenotype is absolutely required forit to progress to Stage D where it responds to pro-inflammatorycytokines/growth factors. Molecules that regulate this transientcellular phase, such as those that either disrupt hyaluronan/hyaladherinor hyaladherin/other protein interactions, make excellent therapeuticand diagnostic targets in a variety of human diseases since thesemolecules will not be expressed in most cells and only transientlyexpressed in diseased tissue. This expression pattern will providetissue specificity and low toxicity to the human body, allowing forchronic use of reagents, a requirement for managing many diseases.

Normal cells surrounded by a normal tissues are quiescent and involvedin the turnover of matrix. Furthermore, consistent with the presentdisclosure, cells do not possess focal adhesions in normal tissue invivo whereas focal adhesions have been observed in diseased tissue.

Transitional stages such as that described above are evident in a widevariety of disease processes, including for example. Parkinson's,Alzheimer's, Arthritis and Osteoporosis. These, as well as other diseaseprocesses which involve transition molecules that remove AP-1restriction from normal cells are discussed in more detail below.

1. Parkinson's

Parkinsonism is a clinical syndrome characterized by a disturbance inmotor functions such as slowness of voluntary movement, diminishedfacial expressions, stooped posture, rigidity and tremor. The diseaseappears later in life. Although little is known on the cause of thedisease, there is substantial evidence indicating that damage to thenigrostriatal dopaminergic system is central to the disease. Thedopaminergic neurons of the substantial nigra project to the striatum innormal brain. In Parkinson's disease, the loss of these neurons resultsin a decrease in striatal dopamine content and this is proportional tothe severity of the motor syndrome. Similar to other brain diseases,there is an increase in glisosis, which involves the recruitment andactivation of glial cells. These cells are recruited as part of therepair process, however, destructive enzymes, reactive oxygen species,cytokines and pro-inflammatory mediators produced by activated glialcells contribute and acerbate the disease.

Thus, within one embodiment methods are provided for treatinginflammatory neurological diseases such as Parkinsons, comprisingadministering to a patient a compound selected from the group consistingof (a) a polypeptide comprising the amino acid sequence BX7B (SEQ IDNO:28) which binds HA: phage display selected peptides that bind HA suchas polypeptides comprising P-15 (Sequence ID No. 70). P-16 (Sequence IDNo. 26); P-32 (Sequence ID no. 71), and GAHWQFNALTVR (Sequence ID No.72); (b) an antibody which binds one of domains D1. D1. D3. D4, or D5 ofRHAMM; (c) a peptide of less than 95 kD or 73 kd, comprising all or aportion of domains D1, D2. D3, D4, or, D5 of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

2 Alzheimer Disease

Alzheimer's disease is clinically manifested as insidious impairment ofhigher intellectual function with alterations in mood and behavior.Later, progressive memory loss and disorientation are observed andeventually, profound disability and death. Alzheimer's disease affects alarge portion of the increasingly aging population with a prevalence ashigh as 47% of those over 85 years old. The total costs required forformal and informal care of AD patients was $67 million in the UnitedStates. Although there is much variability, average life expectancy is8-10 years after dementia onset.

Alzheimer's disease is characterized by the appearance of cerebralextracellular beta-amyloid deposits as senile plaques, intraneuronalneurofibrillary tangles, granulovascular degeneration and amyloidangiopathy. Senile plaques are extracellular lesions comprised ofdegenerating neuronal processes and abnormal deposits of beta-amyloidprotein. Senile plaques range in size from 20 to 200 μm in diameter.Microglia and reactive fibrous astrocytes are enriched in the peripheryof plaques, suggesting the recruitment of cells to the diseased site.These plaques are widely distributed in the cerebral cortex and areconsidered a critical process in the development of the disease.

Neurofibrillary tangles are intraneuronal structures consisting ofpaired helical filaments in which the major constituent is ahyperphosphorylated tau protein, an axonal protein involved inmicrotubule assembly, and neurite regeneration and remodeling. The MAPkinase, ERK, and ubiquitin are also tightly associated with thesehelical filaments and may be directly involved in the stimulation of theMAP kinase pathway. The neurofibrillary tangles represent abnormalorganization of cytoskeletal elements in neurons of patients withAlzheimer's disease.

Other pathological findings associated with Alzheimer's includegranulovascular degeneration, Hirano bodies, neuronal and synaptic loss,and beta-amyloid deposition in the wall of small cortical blood vessels.Although some of these disease processes are found in normal agingbrains, their prevalence is significantly lower than in Alzheimer'sdisease and correlate with the severity of dementia.

Response to neuronal injury is characterized b, the activation of glialcells and the expression of a number of genes that participate in therepair of damaged neurons. Some of those products include thebeta-amyloid precursor protein and neurotrophins. The glial cellrecruitment and responses may compromise neuronal viability by producingcytokines, reactive oxygen species and degradative enzymes. It isgenerally hypothesized that in local neuronal injury, an increasedbeta-amyloid production results in glial cell recruitment and activationwhich results in the production of pro-inflammatory processes and tissuedestruction. Thus, it is most likely the accumulative effects of adefective repair process that results in neuronal cell death and theformation of senile plaques. A potential therapeutic target wouldinclude the inhibition of glial cell recruitment and activation. Thiswould prevent exacerbation of the local inflammation and tissuedestruction.

Thus, within one embodiment methods are provided for treating Alzheimerdisease, comprising administering to a patient a compound selected fromthe group consisting of (a) a polypeptide comprising the amino acidsequence BX7B (SEQ ID NO:28) which binds HA; phage display selectedpeptides that bind HA such as polypeptides comprising P-15 (Sequence IDNo. 70), P-16 (Sequence ID No. 26): P-32 (Sequence ID no. 71); andGAHWQFNALTVR (Sequence ID No. 72): (b) an antibody which binds one ofdomains D1, D2, D3, D4, or D5 of RHAMM; (c) a peptide of less than 95 kDor 73 kd, comprising all or a portion of domains D1, D2, D3, D4, or, D5of RHAMM; and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatthe disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

3. Arthritis and Other Inflammatory Joint Diseases

A number of inflammatory joint diseases have been characterized inhumans based on analysis of signs and symptoms, including for example,rheumatoid arthritis, systemic lupus erythomatosus. Reiter's syndrome,psoriatic arthritis, ankylosing spondylitis, to name just a few.Briefly, rheumatoid arthritis (RA) is the most prevalent type ofinflammatory arthritis which occurs in approximately 1.5% of thepopulation (2). Therefore, the present characterization of humaninflammatory joint disease is based on findings concerning RA. RA ischaracterized by synovial hyperplasia, destruction of articularcartilage and bone, infiltration of lymphocytes and macrophages intosynovial tissues and accumulation of autoantibody immune complexes insynovial fluid. However, the contribution of infiltrating lymphocytecells to the disease process is not clear. Cytokines, such asinterleukin 1 (IL-1) and granulocyte-macrophage colony-stimulationfactor (GM-CSF), are present in increased levels in inflamed joints andplay a major role in the production of metalloproteases including MMP1(collagenase). MMP2 (gelatinase) and MMP3 released by synovial cellswhich are responsible for the destruction of cartilage, IL-1, togetherwith tumor necrosis factor (TNF), also plays a major role inaccumulation of lymphocytes in the joints. Joint inflammation ismediated by plasma and lipid derived mediators, prostaglandin E2 andleukotriene B4.

Gradual destruction of articular cartilage is the most debilitating signof the disease. Cartilage is a connective tissue which consists ofchondrocytes, and extracellular matrix. Collagens and proteoglycans arethe major components of the matrix. Chondrocytes are responsible forpreservation of the integrity of the matrix which mostly depends on thecollagenous network, the majority of which consists of collagen type II.Proteolytic enzymes that degrade the cartilage components aremetalloproteases, which are produced by synovial cells, chondrocytes,neutrophils, and serine proteases derived from neutrophils. Severalfactors can induce the expression of metalloproteases, the most potentbeing secretion of IL-1 by macrophages (4). Tissue inhibitor ofmetalloproteases (TIMP) is a ubiquitous protein and naturalmetalloprotease inhibitor that is present in RA synovial fluid inelevated levels. Another feature of RA is an increase in bone resorptiondue to activation of osteoclasts. It has been shown that monocytederived mediators such a IL-1 and TNF, are responsible for the increasein osteoclastic activity.

In terms of treatment of RA, there are two types of drugs currentlyused: anti-inflammatory drugs, including non-steroid or steroid, whichalleviate the inflammatory process only, and disease modifyinganti-rheumatoid drugs which interfere with the disease process. However,the mechanisms of action of these drugs is mostly unknown.

Osteoarthritis (OA) is a slowly progressive degeneration of thearticular cartilage that manifests in the weight-bearing joints such asthe knees and hips. Osteoarthritis, described as “wear and tear”arthritis, is characterized by narrowing, of the joint owing to the lossof articular cartilage and thickening of the subchondral bone. At alater stage, inflammation of the synovium may occur which plays animportant role in the pathologic process by accelerating the catabolism.All these events lead to nonfunctional and painful joint. The prevalenceand severity of OA increase with age, affecting 80% of the populationafter 55 years of age with higher frequency in women (Altman, 1987). Theprimary cause of OA remains unclear, joint trauma, obesity, bonemicrofractures and aging constitute the risk factors for OA (Altman,1987; Hough et al., 1989).

Although the mechanisms involved in the pathogenesis of cartilagedestruction in OA are not well-characterized, much evidence suggeststhat cytokines may, play an important role. During the progression ofOA, cartilage fragments in the synovial fluid elicit an inflammatoryresponse (Loyau and Pujol, 1990; Pelletier et al., 1991 and 1993). Thisresponse results in enhanced protease and cytokine release and theproduction of reactive oxygen species. The cytokines, including IL-1 andTNFa, can activate MMP synthesis from chondrocytes and synoviocytessetting off a cascade leading to OA (Howell. 1986; Pelletier et al.,1983b). Apart from cytokines, growth factors also have significanteffects on cartilage remodeling.

Thus, within one embodiment methods are provided for treating arthritis(e.g., rheumatoid arthritis or osteoarthritis), comprising administeringto a patient a compound selected from the group consisting of (a) apolypeptide comprising the amino acid sequence BX7B (SEQ ID NO:28) whichbinds HA; phage display selected peptides that bind HA such aspolypeptides comprising P-15 (Sequence ID No. 70), P-16 (Sequence ID No.26): P-32 (Sequence ID no. 71); and GAHWQFNALTVR (Sequence ID No. 72);(b) an antibody which binds one of domains D1, D1, D3, D4, or D5 ofRHAMM; (c) a peptide of less than 95 kD or 73 kd, comprising all or aportion of domains D1, D2, D3, D4, or, D5 of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

4 Osteoporosis

Osteoporosis is a term used to define increased bone porosity of theskeleton resulting from a reduction in bone mass. This disease affectsthe elderly, is particularly prevalent amongst females, and is sometimesa secondary responses to other clinical conditions. Thus, osteoporosismay be primary or secondary, and depending on numerous parameters, canbe localized to a certain bone region or limb, or may involve the entireskeleton. Osteoporosis normally refers to the common primary forms suchas senile and postmenopausal osteoporosis, whereas secondary formsinclude endocrine disorders (hyperparathyroidism, hyperthyroidism,hypothyroidism, acromegaly, Cushing's syndrome, prolactinaoma, Type Idiabetes), neoplasia (multiple myeloma, sarcinomatosis, mast celldisease, thyroid/parathyroid ademo), gastrointestinal disorders(malnutrition, malabsorption, hepatic insufficiency), osteoarthritis andrheumatoid arthritis, drugs (anticoagulants, chemotherapeutics,corticosteroids, lithium), and a number of other non-specific disorders(immobilization or inactivity, pulmonary disease, anemia). Regardless ofthe etiology, the critical loss of bone makes the skeleton vulnerable tofractures and pain. Over 15 million individuals suffer from primaryosteoporosis in the United States and their direct medical costs areover $1 billion annually.

The maximum bone mass is achieved during young adulthood. In normaladults the level of bone mass is determined by genetic factors, diet,physical activity and hormonal state. During adult years and aging thisbone is turned over by a continuous, controlled resorption and formationcycle. In normal individuals, a small deficit in bone mass accrues withevery bone resorption and formation cycle, which can average 0.7% of thetotal bone mass per year. Although there is no doubt that an imbalancein the resorption and formation cycle is responsible for osteoporosis,little is known on the origins of primary osteoporosis. Most of thefocus has been on age-related changes, reduced physical activity andhormonal changes (particularly associated with menopause). It is wellestablished that osteoblasts from the elderly, which are cellsresponsible for bone formation, have reduced biosynthetic potentialrelative to osteoblasts from young adults. In addition, peptides (bonemorphogenic proteins) deposited in the mineralized matrix whichstimulate osteoprogenitor cells and osteoblastic activity are lesseffective with aging. Thus, decreased capacity of bone formationcombined with normal or elevated osteoclastic activity are largelyresponsible for osteoporosis associated with aging and physicalinactivity.

Postmenopausal osteoporosis is characterized by a hormonal dependentaccelerated bone loss. Following menopause, the yearly loss of bone massmay reach 2% of the cortical bone and 9% of the cancellous bone.Estrogen is believed to play an important role in the reduction of boneloss. The estrogen effects are thought to be mediated by cytokines,which are found elevated in osteoporotic bone. It appears that decreasedestrogen levels are capable of inducing cytokines such as IL-1, whichare capable stimulating bone resorption, IL-1 is the most potentstimulator of osteoclast recruitment and activity and thought to play animportant role in bone resorption in post-menopausal osteoporosis. Anumber of genes that are induced by IL-1 (cathepsin K, matrixmetalloproteinases and COX-2) are elevated in osteoporotic bone andproduced by osteoblasts and osteoclasts in vitro. Inhibition ofosteoclast recruitment and activation are key steps in shifting thebalance from resorption to bone formation, resulting in increased bonemass.

Thus, within one embodiment methods are provided for treatingosteoporosis, comprising administering to a patient a compound selectedfrom the group consisting of (a) a polypeptide comprising the amino acidsequence BX7B (SEQ ID NO:28) which binds HA; phage display selectedpeptides that bind HA such as polypeptides comprising P-15 (Sequence IDNo. 70), P-16 (Sequence ID No. 6); P-32 (Sequence ID no. 71); andGAHWQFNALTVR (Sequence ID No. 72): (b) an antibody which binds one ofdomains D1, D2, D3, D4, or D5 of RHAMM; (c) a peptide of less than 95 kDor 73 kd, comprising all or a portion of domains D1, D2, D3, D4, or, D)of RHAMM; and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatthe disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

5. Multiple Sclerosis

Multiple sclerosis is the most common of the demyelinating disorders,having a prevalence of approximately 1 in 1000 persons in most of theUnited States and Europe. Although the etiology of multiple sclerosis(MS) is unknown, genetic, environmental and immunological factors arebelieved responsible for a coordinated attack on myelin. The hallmarklesion in MS is a punched-out area in which the axon is surrounded byastrocytic processes. The accompanying inflammatory reaction ischaracterized by infiltration of lymphocytes, monocytes and macrophagesinto the parenchyma of the central nervous system (CNS), analogous tothe chronic inflammation in other diseases such as arthritis andpsoriasis. Thus, in MS, there is increased inflammatory cell activationand infiltration, increased fibrous astrocyte activation, migration andproliferation, increased production of cytokines and matrixmetalloproteinases, increased demyelination, axonal degeneration andplaque formation.

Thus, within one embodiment methods are provided for treating multiplesclerosis, comprising administering to a patient a compound selectedfrom the group consisting of (a) a polypeptide comprising the amino acidsequence BX7B (SEQ ID NO:28) which binds HA: phage display selectedpeptides that bind HA such as polypeptides comprising P-15 (Sequence IDNo. 70), P-16 (Sequence ID No. 26); P-32 (Sequence ID no. 71); andGAHWQFNALTVR (Sequence ID No. 7); (b) an antibody which binds one ofdomains D1, D2, D3, D4, or D5 of RHAMM: (c) a peptide of less than 95 kDor 73 kd, comprising all or a portion of domains D1, D2, D4, or, D5 ofRHAMM; and (d) a gene delivery vector which expresses antisense RHAMM,or, delivers and expresses any one of (a), (b), or (c), such that thedisease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

6. Inflammatory Dermatosis

Inflammatory dermatological diseases, such as psoriasis, are verycommon, affecting as many as 1 to 2% of the people in the United States.It is often associated with arthritis, myopathy, spondylitic heartdisease and AIDS. Psoriasis is a chronic inflammatory diseasecharacterized by keratinocyte hyperproliferation and a distinctinflammatory pattern that is dependent on the type of psoriasis. Theunderlying pathogenesis involves three predominant and interdependentbiologic processes: inflammation, epidermal hyperproliferation, andaltered differentiation with parakeratosis.

The homeostasis of the epidermis depends on the balance of growthregulatory signals, which appear to be altered in psoriasis. Theepidermis serves a number of important barrier functions against proteinand water loss, entry of microorganisms, physiochemical trauma includingUV. The squamous epithelium undergoes terminal differentiation resultingin an insoluble cornified envelope providing an important barrier.Keratinocyte proliferation takes place in the basal layer and migratethrough the epidermis where differentiation specific proteins such asinvolucrin and keratins are expressed. Normal epidermis represents anormal balance between kaeratinocyte production in the basal layer andcorneocyte shedding at the skin surface. Upon wounding or psoriasis,there are rapid increases in the proliferation of keratinocytes.

Thus, within one embodiment methods are provided for treatinginflammatory dermatosis (e.g., psoriasis), comprising administering to apatient a compound selected from the group consisting of (a) apolypeptide comprising the amino acid sequence BX7B (SEQ ID NO:28) whichbinds HA: phage display selected peptides that bind HA such aspolypeptides comprising P-15 (Sequence ID No. 70). P-16 (Sequence ID No.26): P-32 (Sequence ID no. 71); and GAHWQFNALTVR (Sequence ID No. 72):(b) an antibody which binds one of domains D1, D2, D3, D4, or D5 ofRHAMM; (c) a peptide of less than 95 kD or 73 kd, comprising all or aportion of domains D1, D2, D3, D4, or, D5 of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

7. Inflammatory Bowel Diseases

There is overwhelming evidence that genetic and environmental factorsplay a role in the development of inflammatory bowel diseases (IDB),ulcerative colitis and Crohn's disease. These diseases are chronicrelapsing inflammatory diseases and share many common features ofunknown etiology. Crohn's disease is a granulomatous disease that mayaffect any portion of the gastrointestinal tract from mouth to anus, butmost often involves the small intestine and colon. Ulcerative colitis isa non-granulomatosis disease limited to the colon. These diseases affectapproximately 3 to 6 people per 100,000, but the incidence can varymarkedly between populations.

The clinical manifestations, biochemistry and pathology of IDBdemonstrate that infiltration and activation of inflammatory cells,increased local mucosal responses, overproduction of cytokines anddestructive enzymes are associated Keith the disease process ultimatelyleading to tissue injury. It is not known whether the immune systeminfiltrates the intestine in response to luminal or mucosal antigens orthat local insult or disease results in the expression of adhesionmolecules and chemoattractant cytokines that induce the infiltration ofinflammatory, cells resulting in the immune mediated tissue injury.Regardless of the etiology, there are similarities between the diseaseprocesses in IDB and other chronic inflammatory diseases.

Similar to other inflammatory diseases, there are very high levels ofpro-inflammatory cytokines (IL-1, IL-6, IL-8 and TNF), as well asanti-inflammatory cytokines (IL-4, IL-10 and IL-11) in IBD biopsies. InIBD, there is a disturbed balance between the levels ofpro-inflammatory, cytokines and anti-inflammatory cytokines that favorsthe former. The expression of IL-1, IL-6, IL-8 is increased ininflammatory lesions of patients with IDB (p382-4). These cytokines areproduced by infiltrating inflammatory cells and local epithelial cellsand fibroblasts. It is thought that these imbalances result in increasedexpression of genes such as adhesion molecules, matrixmetalloproteinases and inflammatory mediators that are involved in cellmigration and proliferation, and tissue destruction. Current therapeuticstrategies aim at inhibiting IL-1 and TNF activity.

Thus, within one embodiment methods are provided for treatinginflammatory bowel disease, comprising administering to a patient acompound selected from the group consisting of (a) a polypeptidecomprising the amino acid-sequence BX7B (SEQ ID NO:28) which binds HA;phage display selected peptides that bind HA such as polypeptidescomprising P-15 (Sequence ID No. 70), P-16 (Sequence ID No. 26); P-32(Sequence ID no. 71); and GAHWQFNALTVR (Sequence ID No. 72): (b) anantibody which binds one of domains D1, D2, D3, D4, or D5 of RHAMM; (c)a peptide of less than 95 kD or 73 kd, comprising all or a portion ofdomains D1, D2, D3, D4, or, D5 of RHAMM; and (d) a gene delivery vectorwhich expresses antisense RHAMM, or, delivers and expresses any one of(a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

8. Other Inflammatory Diseases

As described above, there are several classes of molecules and diseaseprocesses that are common to all chronic inflammatory diseases. Theseinclude increased expression of adhesion molecules, cytokines and matrixmetalloproteinases, increased cell proliferation and migration,increased inflammatory cell activation and infiltration, increasedangiogenesis, and increased tissue destruction and dysfunctional matrixremodeling. These disease processes are tightly regulated in normaldifferentiated cells and require the activation of AP-1 transcriptionfactors and AP-1 dependent genes. Since the restriction of AP-1activation in normal cells can be reversed in a controlled fashion bytransition molecules (such as RHAMM), the inhibition of expression,activity and signaling of transition molecules will be usefultherapeutically for not only the diseases described above, but also forother inflammatory diseases such as diabetes mellitus; restenosis;atherosclerosis; systemic lupus erythematosus; emphysema; AIDS; chronicendometriosis; pulmonary, myocardial and hepatic fibrosis; inflammatorypolyradiculoneuropathy; chronic cystitis; acute mastitis; cholecystitis;gastritis; nephritis; hepatitis; bronchial asthma; vasculitis; chronicbronchitis; kidney fibrosis, pericarditis and myocarditis; pancreatitis;peritonitis; prostatitis; septic shock; periodentitis, thyroiditis;retinopathy.

Thus, within one embodiment methods are provided for treating the abovedescribed treating diseases (e.g., lupus, diabetes mellitus, or, kidneyfibrosis), comprising administering to a patient a compound selectedfrom the group consisting of (a) a polypeptide comprising the amino acidsequence BX7B (SEQ ID NO:28) which binds HA; phage display selectedpeptides that bind HA such as polypeptides comprising −15 (Sequence IDNo. 70), P-16 (Sequence ID No. 26); P-32 (Sequence ID no. 71); andGAHWQFNALTVR (Sequence ID No. 72); (b) an antibody which binds one ofdomains D1, D2. D3, D4, or D5 of RHAMM; (c) a peptide of less than 951kD or 731 kd, comprising all or a portion of domains D1, D2, D3, D4, or.D5 of RHAMM; and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatthe disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

9. Wound Healing and Responses to Injury

Wound healing responses to injury involve a complex series of cellularand inflammatory processes resulting in the deposition of connectivetissues and its remodeling, into abnormal tissue or scarring. Theunderlying mechanisms of wounding or injury responses involve theinduction of an acute inflammation, production of cytokines and growthfactors, regeneration of parenchymal cells, migration, proliferation anddifferential of parenchymal and connective tissue cells, synthesis ofextracellular matrix proteins, angiogenesis and fibrosis, and remodelingof connective tissues. In addition, these healing processes are commonin a variety of clinical areas such as scarring from surgical incisions,wounds or various derma inflammatory diseases, restenosis followingangioplasty, vascular grafts, stroke and surgical adhesions.Interference of processes that induce abnormal tissue deposition andremodeling will enhance an orderly wound or injury repair resulting inthe development of normal functional tissue. Since transition molecules,such as RHAMM, regulate a number of the diseased processes in woundhealing and the transformation of normal to diseased cells, it is likelythat agents which inhibit the function of transition molecules would beuseful therapeutically for the treatment of restenosis followingangioplasty, vascular grafting, ballooning or any other type of injuryto the vascular system, stroke, surgical incisions, burns, wounds,inflammatory skin diseases, and surgical adhesions.

The simplest form of wound repair or healing is observed following aclean surgical incision. The incision causes a limited amount of tissuedisruption, which results in responses by epithelial cells andconnective tissue cells, as well as infiltration of inflammatory cells.Immediately following the incision, the incision space is bathed withblood, containing fibrin and blood cells that clots and leads to theformation of a scab that covers the wound. The initial process involvesthe response of local basal cells in the production of cytokines andother pro-inflammatory mediators, and infiltration of neutrophils. Thebasal cells become mitotic and produce matrix, resulting in thethickening of the epidermis. This is followed by the migration of theepithelial cells along the cut margins and depositing basement membraneunderneath the scab. The neutrophils are replace by macrophages andgranulation tissue is progressively laid down containing collagenfibrils vertically oriented rather than oriented in fashion that wouldenhance bridging the incision space. Epithelial cell proliferation andmigration continues, as well as tissue thickening. Neovascularizationreaches maximal levels and the surface cells differentiate and producenormal epidermal architecture. The last stages of incision healinginvolve the disappearance of all inflammatory cells, edema and increasedvascularization, as well as accumulation of normal collagen fibrils andstrengthening of tissue.

In cases where there are more extensive surface wounds such as burns,abscess formation, inflammatory ulceritis, the reparative process isalso more extensive. The larger tissue defects have greater cell loss,more fibrin and more inflammation, increased amounts of granulationtissue and wound contraction involving myofibroblasts. Regardless of thewound, the mechanisms of responsible for the processes of healingdescribed above are similar. Wound healing is ultimately regulated bygrowth factors and cytokines that balance matrix synthesis anddegradation locally. Collagen synthesis is a key component of woundhealing and provides the tensile strength required closing of theincision. The type of collagen produced is dependent on the tissuerepaired, and changes in the type of collagen may lead to dysfunctiontissue. Collagen synthesis is stimulated early in tissue repair byfactors such PDGF, FGF, and TGF. On the other hand, degradation ofcollagen fibrils and other matrix molecules are also important. Thedegradative enzymes involved during wound healing include matrixmetalloproteinases, neutrophil elastase, cathepsin G, kinins, plasminand other enzymes. Inflammatory and local cells produce these enzymes.Degradation may aid in the remodeling of the connective tissue repair.If the inflammatory destructive processes are suppressed, then it ismore likely to achieve a more rapid formation of the connective tissuesand decrease the accumulation of scar tissue.

One type of wound healing occurs in surgical adhesions. Briefly,surgical adhesion formation is characterized by abnormal adherence andscar formation between two adjacent tissues that occur most oftenfollowing surgery. Adhesions are a major cause of surgical therapy andcan result in bowel or urethral obstruction. Surgical adhesions arethought to be an inflammatory response to surgical trauma. Local tissuesand inflammatory cells produce and secrete pro-inflammatory cytokineswhich increase vascular permeability, inflammatory cell infiltration,cellular migration and proliferation, and the laying down of matrixbetween just-neighboring tissues. The accumulation of fibroblastsresults in the accumulation of matrix and eventual adhesion of the twotissues. In theory, any agent that inhibits the inflammatory responseand tissue remodeling would prevent the formation of surgical adhesions,particularly if these agents can be administered locally.

Thus, within one embodiment methods are provided for treating theaforementioned diseases associated with wounds/wound healing, comprisingadministering to a patient a compound selected from the group consistingof (a) a polypeptide comprising the amino acid sequence BX7B (SEQ IDNO:28) which binds HAA: phage display selected peptides that bind HAsuch as polypeptides comprising P-15 (Sequence ID No. 70), P-16(Sequence ID No. 26); P-32 (Sequence ID no. 71); and GAHWQFNALTVR(Sequence ID No. 72); (b) an antibody which binds one of domains D1, D2,D3, D4, or D5 of RHAMM; (c) a peptide of less than 95 kD or 73 kd,comprising all or a portion of domains D1, D2, D3, D4, or, D5 of RHAMM;and (d) a gene delivery vector which expresses antisense RHAMM, or,delivers and expresses any one of (a), (b), or (c), such that thedisease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

10. Restenosis/Stenosis following Therapeutic Interventions in VascularDisease: Angioplasty, Stent Insertion and Vascular Replacement/Grafts

Vascular diseases such as atherosclerosis are a leading cause of deathand disability in the developed world. Several therapeutic interventionshave been developed to treat vascular diseases such as atherectomy,balloon angioplasty, insertion of stents, and insertion of arterial andvenous grafts. For example, over 300,000 procedures of percutaneoustransluminol coronary angioplasty are performed in the United States peryear. Although these interventions are less costly and less invasive tothe patient, there are a number of morphological changes and diseasestates produced in response to injury that are introduced by these newmodes of therapy, namely restenosis.

Restenosis is characterized by thickening of the blood vessel wall inresponse to injury that progresses until full occlusion of the vessel.Despite the significant advance made in these therapies, chronicrestenosis of the dilated lesions occur in 30 to 50% of the cases,remaining a serious and frequent problem. Furthermore, eventuallystenosis occurs in virtually all grafted vessels. Restenosis has beensuggested to represent an exaggerated healing response to local injury,in which smooth muscle cells in the media migrate to and proliferate inthe intima. Local production of cytokines and growth factors by localcells and inflammatory cells results in abnormal matrix deposition andremodeling. There are number of underlying mechanisms which can play arole in the induction of this disease. An injury to the endothelial celllayer will expose blood vessel layers to serum components and platelets,initiating a wound healing process. Factors released locally lead toincreased cell proliferation and increased expression of matrixmetalloproteinases required for cell proliferation and migration. Thesecells accumulate in the intima and form lesions that eventually blockthe vessel. Utilizing the therapeutic compositions provided herein,blocking the activation of smooth muscle cells and inhibition of theirmigration and proliferation in response to injury can be utilized totherapeutically treat stenosis and restenosis.

Thus, within one embodiment methods are provided forinflammatory/proliferative diseases associated with surgical proceduresor intervention (e.g., restenosis, stenosis, medical implants and thelike), comprising administering to a patient a compound selected fromthe group consisting of (a) a polypeptide comprising the amino acidsequence BX7B (SEQ ID NO:28) which binds HA; phage display selectedpeptides that bind HA such as polypeptides comprising −15 (Sequence IDNo. 70), P-16 (Sequence ID No. 26); P-32 (Sequence ID no. 71); andGAHWQFNALTVR (Sequence ID No. 72); (b) an antibody which binds one ofdomains D1, D2, D3, D4, or D5 of RHAMM; (c) a peptide of less than 95 kDor 73 kd, comprising all or a portion of domains D1, D2, D3, D4, or, D5of RHAMM; and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatthe disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally. In addition, within certainembodiments the compounds described herein may be administered byballoon catheter, or, delivered from a stent which is adapted to releasethe desired compound.

11. Atherosclerosis and Related Diseases: Myocardial Infarction andStroke

Cardiovascular disease is a serious problem and accounts for 44% of themortality in the USA. Atherosclerotic cardiovascular disease isgeneralized process that involves the brain, heart and peripheralarteries. Atherosclerosis is characterized by intimal thickening causedby the accumulation of cells, infiltration of inflammatory cells,lipids, and connective tissues that can lead to cardiac and cerebralinfarction (such as heat attack and stroke). Although the role ofinjurious stimuli is not known, the responses of the endothelial cellsand the adaptive changes within the intima are critical in vascularremodeling leading to atherosclerotic plaques. Endothelia cells,monocytes and smooth muscle cells express biologically active moleculessuch as adhesion molecules, cytokines, coagulation and fibrinolyticfactors, metalloproteinases and vasoactive substances that contribute toatherogenesis and thrombosis. It is thought that atherosclerotic lesionsdevelop by (1) invasion of artery wall by inflammatory, cells,particularly monocytes: (2) smooth muscle cell migration, proliferation,and synthesis of matrix molecules; (3) intracellular lipoprotein uptakeand lipid accumulation. Briefly, inflammatory cytokines induce theproduction of adhesion molecules resulting in inflammatory cellinfiltration and responses. Activated smooth muscle cells migrate inresponse to local injury and produce large amounts of matrix and expresslipoprotein scavenger receptors and can become involved in a generalizedimmune reaction. Occlusion of the artery leads to a series of clinicalcomplications such as myocardial infarction and stroke. Prevention ofinflammatory cell infiltration, production of matrix metalloproteinases,cell proliferation and migration will reduce smooth muscle cell andmatrix accumulation, and inhibit vessel occlusion.

Thus, within one embodiment methods are provided for treating theabove-noted atherosclerotic diseases, comprising administering to apatient a compound selected from the group consisting of (a) apolypeptide comprising the amino acid sequence BX7B (SEQ ID NO:28) whichbinds HA; phage display selected peptides that bind HA such aspolypeptides comprising P-15 (Sequence ID No. 70), P-16 (Sequence ID No.26); P-32 (Sequence ID no. 71); and GAHWQFNALTVR (Sequence ID No. 72);(b) an antibody which binds one of domains D1, D2, D3, D4, or D5 ofRHAMM; (c) a peptide of less than 95 kD or 73 kd, comprising all or aportion of domains D1, D2, D3, D4, or, D5 of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

12. Tissue Transplantation

The increasing use of transplantation for bone marrow, renal, pulmonary,cardiovascular and hepatic disorders has generated a series of clinicalcomplications. In addition, with recent advances in tissue engineering,there is considerable potential that skin, cartilage, bone and manyother tissues will be transplanted in the future. In many casestransplantation is the only form of treatment. For example, lungtransplant is the only effective treatment of terminal lung diseasessuch as idiopathic pulmonary fibrosis, primary pulmonary hypertension,emphysema, and cystic fibrosis. The same is true for specific renal,hepatic and heart diseases. There are three major complications in thetransplantation of organs: (1) host versus graft disease; (2)non-immunological damage; and (3) infection. Acute and chronic rejectionis a significant problem where the host immune system invades the donororgan. This inflammatory response and mononuclear cell infiltrates aretreated with immunosuppressive drugs with some success. However, thesedrugs can be very toxic and result in other clinical complications. Thenon-immunological damage from preservation injury results ininflammation and tissue damage. The role of infection can be treatedwith antibiotics. The disease processes involved in organ rejection aresimilar to other inflammatory diseases.

Disease intervention with devices has increased significantly over thepast decade. These include the use of devices for hip and kneereplacements, cardiovascular stents, esophageal stents, vascular wraps,bone crafts, venous and arterial grafts, many others. A common problemwith the use of these devices is an inflammatory reaction to particlesproduced from the device or loosening of the device or injury caused bythe local application of the device. It would seem likely that systemicor local application of the inflammatory response and local tissuereaction to the devices would inhibit this problem.

Thus, within one embodiment methods are provided for treating patientsundergoing tissue or cell transplation, comprising administering to apatient a compound selected from the group consisting of (a) apolypeptide comprising the amino acid sequence BX7B (SEQ ID NO:28) whichbinds HA; phage display selected peptides that bind HA such such aspolypeptides comprising P-15 (Sequence ID No. 70), P-16 (Sequence ID No.26); P-32 (Sequence ID no. 71); and GAHWIQFNALTVR (Sequence ID No. 72);(b) an antibody which binds one of domains D1, D2, D3, D4, or D5 ofRHAMM; (c) a peptide of less than 95 kD or 73 kd, comprising all or aportion of domains D1, D2, D3, D4, or, D5 of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

13. Cancer and Metastases

Cancer is a generic term representing a collection of diseases arisingfrom mutations of key molecules that regulate cell proliferation,invasion, and metastasis. A representative cancer for which the keymutations are known is exemplified by colorectal cancer. This canceroriginates as a benign growth as a result of a mutation in a gene termedAPC. Mutation of three additional molecules are required for this benigngrowth to progress to a rapidly proliferating and invasive tumor. Aplethora of mutations arises within the tumor as it progresses and theseenhance the ability of the mutant tumor cells to attract normalendothelial cells to migrate into the growing tumor and form new bloodvessels, a process known as angiogenesis. As angiogenesis proceeds andas mutations affecting the ability of tumors to respond to growthfactors accumulate, subsets of tumor cells develop the capacity toinvade blood vessels as well as lymphatics and to metastasize.

The ability of tumor cells to metastasize involves deregulation viaoverproduction or mutation of genes that allow cells to invade out ofthe tissue of origin, survive in a contact-independent manner, escapeimmune recognition, lodge at a distant site, then invade to a suitableplace within the new tissue and grow there. The molecules that arecommonly involved in tumor initiation, progression and metastasisinclude adhesion molecules, growth factor receptors, factors regulatingthe cytoskeleton, master switches regulating cell cycle, proliferationrepressor genes, proteases and transcription factors.

Although our understanding of master switches, proliferation repressors,growth factors and proteases is quite well developed and pre-clinicaland clinical approaches to targeting these molecules, particularlyproteases have been developed, very little is known about the molecularcharacteristics of the invasive phenotype. The invasive tumor phenotypeis predicted to be similar to the transitional phenotype noted for theabove diseases and to be characterized by a propensity to forminvadapodia or podosomes to release proteases and to express transitionmolecules that permit and prepare a cell to invade, move, and ultimatelyrespond to growth factors and cytokines in a focal adhesion-dependentmanner. It is likely that molecules required for generating thisphenotype are also expressed transiently in tumor cells since they maybe only temporarily required and permanent expression would notnecessarily be advantageous. Thus, it is predicted that transitionalmolecules defining an invasive phenotype would appear in a subpopulationof tumor cells in a given tumor. A transient nature is likely one reasonthat markers of invasive phenotype have been so elusive to define.However, the ability of most tumors to kill is directly related to theircapacity to invade and ultimately to metastasize. Therefore,identification of transient molecules is key for diagnosis, prognosis,adjuvant treatment or therapeutic treatment of a variety of cancersincluding: head and neck tumors (lip, oral cavity, auropharynx,nasopharynx, hypopharynx, larynx, glottis, supraglottis, subglottis,maxillary sinus, major salivary gland, lung, esophageal, gastric,colorectal cancer, anal, pancreatic liver, gall bladder, extrahepaticbile duct cancer, breast cancer, gynecologic cancers (cervix,endometrium, ovary, cancer of the uterine body, vaginal, vulvar,gestational trophoboblastic), testicular, urinary tract (renal, urinarybladder, penile, urethral, prostatic) neurologic, endocrine skin (basalcell and squamous cell melanoma) sarcomas, blood (leukemia, lymphoma)childhood neoplasm's (leukemia, lymphoma, neuroblastoma, Wilms' tumorrhabdomyosarcoma, Ewing's sarcoma, retinoblastoma) mediastinum, thymicgerm cell, retroperitoneal, cardiovascular tumors, mastocytosis,carcinosarcomas, adenoid cystic carcinoma, dental tumors olfactory,neuroblastoma, paraganglioma.

With regard to transitional molecules involved in proliferative cancers,the present invention shows that RHAMM is highly overexpressed insubsets of cells in primary breast cancer tissue and this overexpressionis prognostic of lymph node metastasis and poor outcome. Furthermore,RHAMM is shown to regulate ERK activation, a key player in AP-1activation. ERK is also shown to regulate cell locomotion, a keybehavior required for cell invasion into lymph nodes and is required forthe invasion of tumor cells both in vitro and in transgenic models ofbreast cancer. Furthermore, CD44 is required for efficient signalingthrough her2/neu, an oncogene strongly implicated in regulating lymphnode metastasis of breast cancer cells. Finally, HA promotes theexpression of podosomes in invasive cancer cells and podosome formationis one important characteristic of the transitional phenotype. Inaddition, and consistent with this observation. HA promotes the invasionof these cells into collagen gels in vitro.

Thus, within one embodiment methods are provided for treating cancer andother metaseses, comprising administering to a patient a compoundselected from the group consisting of (a) a polypeptide comprising theamino acid sequence BX7B (SEQ ID NO:28) which binds HA; phage displayselected peptides that bind HA such as polypeptides comprising −15(Sequence ID No. 70), P-16 (Sequence ID No. 26); P-32 (Sequence ID no.71); and GAHWQFNALTVR (Sequence ID No. 72); (b) an antibody which bindsone of domains D1, D2, D3, D4, or D5 of RHAMM; (c) a peptide of lessthan 95 kD or 73 kd, comprising all or a portion of domains D1, D2, D3,D4, or, D5 of RHAMM; and (d) a gene delivery vector which expressesantisense RHAMM, or, delivers and expresses any one of (a), (b), or (c),such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

14. Chronic and Acute Respiratory Distress Syndrome:

Due to injury of the lung such as occurs in premature birth andconsequent positive pressure breathing measures as well as in adultsfollowing accidents or chemotherapy, the lung is injured and macrophagesand neutrophils accumulate as within the lung eventually destroying typeII aveolar cells that produce surfactant proteins required formaintenance of positive pressure following lung expansion. As a result,lungs are poorly functional and patients become cyanotic and breatherapidly. This syndrome ends in death. Clinical indications characterizedby lung inflammation include emphysema, asthma, cystic fibrosis,new-born lung disease involving chronic respiratory distress syndrome,and the acute respiratory distress syndrome that affects accidentvictims. Local inflammatory responses that recruit macrophages into thelung result in destruction of alveolar type II cells, which make thesurfactant responsible for normal lung inflation. The infiltration ofmacrophages and abnormal local tissue responses result in further tissuedestruction and disease. This pathological sequence results in improperlung expansion. As described in more detail herein, reagents thatinhibit transitional proteins prevent massive accumulation of whitecells that result in this syndrome and prevent the development of asurfactant deficit in the lung.

Thus, within one embodiment methods are provided for treating chronicand acute distress syndromes, comprising administering to a patient acompound selected from the group consisting of (a) a polypeptidecomprising the amino acid sequence BX7B (SEQ ID NO:28) which binds HA;phage display selected peptides that bind HA such as polypeptidescomprising −15 (Sequence ID No. 70), P-16 (Sequence ID No. 26); P-32(Sequence ID no. 71); and GAHWQFNALTVR (Sequence ID No. 72); (b) anantibody which binds one of domains D1, D2, D37 D4, or D5 of RHAMM; (c)a peptide of less than 95 kD or 73 kd, comprising all or a portion ofdomains D1, D2, D3, D4, or, D5 of RHAMM; and (d) a gene delivery vectorwhich expresses antisense RHAMM, or, delivers and expresses any one of(a), (b), or (c), such that the disease is treated.

The polypeptides, antibodies, or, vectors may be delivered to thepatient by a variety of routes, including for example; systemically,intravenously, intramuscularly, and orally.

15. Diabetes Mellitus

Diabetes mellitus is a group of diseases characterized by high glucoseresulting from defects in insulin secretion, insulin action, or both.Diabetes mellitus can be associated with serious complications such asheart disease, stroke, kidney disease, nervous system disease, blindnessand complications in pregnancy.

Type I diabetes mellitus, also referred to as insulin dependent diabetesmellitus (IDDM), develops most often in children and young adults over ashort period of time. About 30-40% of diabetic children eventuallydevelop nephropathy. Type II diabetes mellitus usually develops inadults. Risk factors include obesity and family history of diabetes. Thesymptoms usually develop gradually and are not as noticeable as in TypeI diabetes.

Type I diabetes mellitus is an autoimmune disorder, the onset of whichresults from a well characterized insulitis. During this condition theinflammatory cells are apparently specifically directed against theinsulin producing beta cells of the pancreatic islets. The destructionof pancreatic beta cells by invading leukocytes result in deteriorationof the insulin-dependent homeostasis.

The inflammatory cascade is a complex process that involves triggeringof the immunological response, release of chemokines, cytokines and atoxic agents by the activated cells, up-regulation of cell surfaceadhesion molecules and transendothelial cell migration. Although thetriggering mechanism of IDDM remains elusive, it is clear that theentire process depends on the migration of inflammatory cells into thepancreatic islets and their interaction with matrix.

Within one embodiment methods are provided for treating or preventingdiabetes mellitus, comprising administering to a patient a compoundselected from the group consisting of (a) a polypeptide comprising theamino acid sequence BX7B (SEQ ID NO:28) which binds HA; phage displayselected peptides that bind HA such as polypeptides comprising P-15(Sequence ID No. 70), P-16 (Sequence ID No. 26); P-32(Sequence ID no.71); and GAHWQFNALTVR (Sequence ID No. 72): (b) an antibody which bindsone of domains D1, D2, D3, D4, or D5 of RHAMM; (c) a peptide of lessthan 95 kD or 73 kd, comprising all or a portion of domains D1, D2, D3,D4, or D5 of RHAMM; and (d) a gene delivery vector which expressesantisense RHAMM, or, delivers and expresses any one of (a), (b), or (c),such that the disease is treated. Within certain embodiments of theinvention, the compounds described herein may be administered before,during, or subsequent to islet-cell transplantation. Within otherrelated embodiments, the above-described compounds may be utilized totreat related diseases, including for example, obesity.

The polypeptides, antibodies, or vectors may be delivered to the patientby a variety of routes, including for example, systemically,intravenously, intramuscularly, and orally.

Pharmaceutical Compositions

As noted above, the present invention also provides a variety ofpharmaceutical compositions, comprising one of the above-describedmolecules with a pharmaceutically or physiologically acceptable carrier,excipients or diluents. Generally, such carriers should be nontoxic torecipients at the dosages and concentrations employed. Ordinarily, thepreparation of such compositions entails combining the therapeutic agentwith buffers, antioxidants such as ascorbic acid, low molecular weight(less than about 10 residues) polypeptides, proteins, amino acids,carbohydrates including glucose, sucrose or dextrins, chelating agentssuch as EDTA, glutathione and other stabilizers and excipients. Neutralbuffered saline or saline mixed with nonspecific serum albumin areexemplary appropriate diluents.

In addition, the pharmaceutical compositions of the present inventionmay be prepared for administration by a variety of different routes(e.g., systemically, orally rectally, intravenously, intramuscularly,ocularly, or, topically). Further within other embodiments the compoundsor compositions provided herein may be admixed with other carriers(e.g., polymers), and implanted on or contained within devices which aredesigned to release such compounds. Within further embodiments, thecompounds may be delivered under radioscopic or other visual guidance toa desired site (e.g., outside the lumen of a desired vessel, or outsideof an organ, or, tissue to be treated).

As should be readily evident, the compounds or compositions of thepresent invention should be administered sufficient to have the desiredtherapeutic outcome. As an example, it is generally desirable toadminister between a total of 1 ng of the desired compound, and up to 80mg/kg. Within certain embodiments, the dosage will be adjusted for thetherapeutic regimen desired (e.g., from 1 ug/kg to 1 mg/kg). Withinother embodiments the dosage for local administration may range from 1to 100 ug/ml (2.5 ng/kg to 80 mg/kg), and for systemic administrationfrom 1 ng/kg to 10 mg/kg. Further, the dosage can be adjusted based uponthe desired route of treatment, e.g., a smaller dose may be given ifapplied locally or topically, whereas a larger dose may be given if thecompound is administered systemically. Further, the dosage may vary withthe desired regimen (e.g., daily, weekly, or monthly).

In addition, pharmaceutical compositions of the present invention may beplaced within containers, along with packaging material which providesinstructions regarding the use of such pharmaceutical compositions.Generally, such instructions will include a tangible expressiondescribing the reagent concentration, as well as within certainembodiments, relative amounts of excipient ingredients or diluents(e.g., water, saline or PBS) which may be necessary to reconstitute thepharmaceutical composition.

The following examples are offered by way of illustration, and not byway of limitation.

EXAMPLES Example 1 Requirement for Focal Adhesions for MaximalActivation of ERK Kinase in Response to Growth Factors

In disease or injury, mediators such as cytokines, growth factors andgenetic mutations activate a myriad of responses leading in increasedexpression of AP-1 dependent genes (FIG. 1). These genes are requiredfor cell proliferation, migration, inflammation, tissue destruction andabnormal tissue remodeling. The activation of the AP-1 pathway occursthrough the activation of the MAP kinase. The present inventiondiscloses that in normal cells the activation of the AP-1 pathway bycytokines and other mediators is restricted, and thus genes involved indisease cannot be induced significantly. Further this restriction is aresult of the lack of ERK-1 activation in normal cells (FIG. 2). Normalcells must undergo a series of transitional stages to form a diseasedstate cell containing focal adhesions and is then responsive toinflammatory mediators. Transition stage cells provided by the presentinvention constitutively form podosomes and are unable to establishfocal adhesions. Sustained formation of podosomes leads to the formationof focal adhesions and results in a diseased state (FIG. 3). The presentinvention further discloses a requirement for focal adhesions formaximal activation of erk kinase in response to growth factors andcytokines. Cellular response-to-injury processes including growth factormediated responses which lead to cellular proliferation, migration,production of destructive enzymes and abnormal tissue remodeling arecharacterized by a maximal activation of the erk kinase signalingpathway. To demonstrate that this response requires the presence offocal adhesions, the response to IL-1 induction of erk kinase signalingwas measured in cells grown under conditions permitting or preventingthe formation of focal adhesions.

Cells were either plated without serum on culture dishes precoated at 4°C. overnight with 25 μg/ml fibronectin which permits formation of focaladhesions or with 100 μg/ml poly-l-Lysine which prevents formation offocal adhesions. Formation of focal contacts was detected by positiveimmunofluorescence of the marker, protein, vinculin. Activation of erkkinase signaling in comparison to other MAP kinase signaling pathwaysregulated by growth factors was estimated by detection of proteinsphsophoryalted by components of the differing signaling cascades.Phosphorylation of myelin basic protein (MBP) is an indicator of erkkinase signaling, phsophorylaltion of GST-c-jun is an indicator of jnksignaling, and phsophoryaltion of GST-ATF2 is an indicator of p38 kinasesignaling cascade. Results of this analysis is shown in FIG. 4.

More specifically, FIG. 4B shows that cells plated onto fibronectin (FN)are able to form focal contacts as detected by positiveimmunofluorescence for the marker protein vinculin. Cells that aremaintained on a non-physiological yet adhesive substratum poly-L-lysine(PL), attach but do not form focal contacts (4A). FIG. 4C shows thatnormal quiescent phase cells plated onto fibronectin substrata whichmake focal contacts are able to activate the erk kinase cascade asindicated by the phosphorylation of myelin basic protein (MBP) inresponse to the cytokine IL-1 (lane 2). These same cells plated ontopoly-L-lysine do not make focal contacts and are unable to activate erkas detected by MBP phosphorylation (lane 1). However, cells plated ontofibronectin (“FN”) or in suspension are equally able to activate theother map kinases, jnk or p38 (lanes 1 and 2). FIG. 4C also shows, thatnormal cells in the absence of focal adhesions, when plated ontofibronectin or grown in suspension (SP), are restricted in their abilityto activate erk in response to IL-1 in comparison to disease cellscontaining focal adhesions, but able to activate the other MAP kinases,jnk and p38 (lanes 3 and 4). These results indicate that responsivenessof the erk kinase cascade is restricted in transition stage cells butthat the erk kinase cascade becomes maximally active when focal contactsare made as occurs upon entry of cells into a post-transitional stagethat is fully responsive to growth factor stimulation, as indicated inFIG. 3.

Northern analysis was used to further demonstrate IL-1 induction of theAP-1 transcriptional activator, c-fos, by cells able to form focalcontacts, IL-1β was added to cells grown either on FN or PL, then RNAwas isolated and analyzed by Northern blotting for levels of c-fos mRNA.FIG. 4D shows a Northern analyses of cells plated on fibronectin or PLand incubated with 20 ng/ml of IL-1β. 20 ng/ml IL-1β was able to inducec-fos expression in cells grown on FN (cells with focal adhesions) butnot in cells grown on PL (in the absence of focal adhesions). Blots werefirst probed for c-fos mRNA expression, stripped, and then reprobed withcontrol radiolabeled GAPDH cDNA to assess equality of RNA loading.

FIG. 4E shows that the level of AP-1 activated in response to IL-1induction requires the ability to make focal adhesions (cells grown onPL which are unable to form focal adhesions have reduced levels of AP-1induction relative to cells grown on fibronectin).

More specifically, to further demonstrate a requirement of focaladhesions for full IL-1 induction, the amount of the transcriptionalfactor AP-1 binding induced in response to IL-1 stimulation wasanalyzed. The level of DNA binding to an AP-1 oligonucleotide wasmeasured in nuclear extracts from cells either grown on fibronectin orpoly-1-Lysine coated dishes in medium without serum. Briefly, tissueculture dishes were precoated with 25 μg/ml fibronectin or 100 μg/mlpoly-1-Lysine as before and washed twice with PBS before use. Cells werethen incubated under starving condition for 6 h. media were removed andfresh serum-free medium containing IL-1 (20 ng/ml) was added to thecells for 4 h.

For the preparation of nuclear extracts, cells were washed twice withPBS (phosphate-buffered saline) and lysed with 1 ml buffer 1 (10 mMTris-Cl, pH 7.5, 10 mM NaCl, 3 mM MgCl₂, 0.5% Nonidet P-40, 0.5 mMphenylmethylsulfonyl fluoride (PMSF). Cells were scraped into aneppendorf tube and put on ice for 10 min. The nuclei were collectedafter centrifugation at 5000 rpm for 10 min. Nuclear proteins wereprepared by resuspending the nuclei in buffer 2 (20 mM Hepes, pH7.9, 5mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 300 mM NaCl, 20% glycerol, 0.5 mMPMSF), after centrifugation at 14,000 rpm for 10 min, supernatant washarvested. Double-stranded AP-1 oligonucleotide (Santa Cruz Biotech,Inc) was end-labeled with [γ-³²P] ATP (DuPont NEN) using T4polynucleotide kinase (Pharmacia). Labeled probe was separated from freenucleotide through a Sephadex G-50 mini-spin column (Pharmacia).DNA-protein binding was performed by mixing 10 μg of nuclear extractwith ³²P-labeled double-stranded AP-1 consensus oligonucleotide in atotal volume of 20 μl containing 20 mM Hepes, pH 7.9, 1 mM MgCl₂, 4%Ficoll, 0.5 mM DTT, 50 mM KCl, 1 mM EDTA, 2 μg poly(dI·dC) and 1 mg/mlBSA for 45 min on ice. The DNA protein complex was separated on a 4%native polyacrylamide gel using 0.5× Tris-borate-EDTA buffer at 150 V.Gels were then dried and autoradiographed.

Example 2 RHAMM Overexpression is Associated with Increased ERK KinaseActivation and AP-1 Activation

As noted above, expression of transitional molecules such as RHAMMresults in the initiation of cell transformation from a normal state toa diseased state. RHAMM is believed to play a role in the initialactivation of ERK pathway thus removing the ERK restriction found innormal cells. This activation leads to the expression of c-fos and c-junresulting in the AP-1 activation and induction of AP-1 dependent genesinvolved in many of the disease processes associated with inflammation,degenerative and proliferative diseases (FIG. 5).

Cells that overexpress a hyaladherin such as RHAMM in response to stressor during proliferation exhibit elevated activation of erk kinasesignaling activity as shown in FIG. 6. Erk kinase activation isstimulated directly by overexpression of a hyaladherin such as RHAMM.Briefly, the cell line LR21 was constructed by transfecting normalquiescent parental 10T1/2 cells with a vector expressing a RHAMMv4polypeptide. Cells that overexpress RHAMM show increased erk activationas indicated by phosphorylation of the MAP kinase activated myelin basicprotein (MBP), p44 ERK1 and p42 ERK2, and by increased AP-1 bindingactivity.

FIG. 6A illustrates that MAP kinase activity in quiescent 10T1/2 cellsis reduced relative to the levels present in RHAMM transfected LR21cells. Cells were growth in DMEM with 10% FBS, cell monolayers werewashed three times with PBS and total cellular extracts were prepared ina buffer containing 25 mM Hepes, pH 7.7, 100 mM NaCl, 2 mM MgCl₂, 0.2 mMEDTA, 0.5% Triton X-100, 0.5 mM DTT, 20 mM β-glycerophosphate, 0.1 mMsodium orthovanadate, 0.5 μg/ml leupeptin, 100 μg/ml PMSF. Cellularlysates of 100 μg total protein were incubated with anti-ERK-1 antibodyconjugated agarose (ERK(C-14). Santa Cruz Biotech. Inc),immuno-complexes were washed twice with the above lysis buffer and twicewith kinase buffer (20 mM Hepes, pH 7.7, 10 mM MgCl₂, 2 mM MnCl₂, 2 mMDTT and 25 μM ATP). ERK2 activity was determined by in vitro kinaseassay using 2 μg substrate MBP and 1 μCi [γ-³²P] ATP in 20 μl of kinasebuffer. After incubation at 30° C., for 20 min, the reactions wereterminated with Laemmli buffer, proteins were separated by SDS-PAGE andthe gels were dried and autoradiographed.

The amount of ERK2 and phosphorylated MAPK was detected from the totalextracts by western blot using an ECL chemiluminescence system. Inbrief, lysates of 25 μg total protein were resolved by 10% SDS-PAGE andtransferred onto nitrocellulose membrane (BioBlot, Costar) usingTrans-Blot® Semi-Dry Electrophoretic Transfer Cell (BioRad) with atransfer-blotting buffer containing 20 mM Tris, 150 mM glycine, 0.01%SDS and 20% methanol. The filters were blocked for non-fat skim milk inTBS-T (20 mM Tris, pH 7.5, 150 mM NaCl and 0.1% Tween 20) at 4° C.overnight. The membranes were then probed with phospho-specificanti-p44/p42 MAP kinase antibody (New England BioLabs, Inc.) byincubation at room temperature for 1.5 h. After washing three times withTBS-T for 30 min, blots were incubated with horseradish peroxidaseconjugated anti-rabbit antibodies (NEB) for 1 h. The filters were washedthree times for 30 min and visualized on X-ray film using thechemiluminescence detection method (NEB).

FIG. 6B illustrates that AP-1 DNA binding activity is stimulated in LR21cells relative to parental 10T1/2 cell. Parental 10T1/2 cell and LR21cells were grown in DMEM with 10% FBS. Cells were then starved in themedium without serum for 8 h. Cells were washed twice with PBS(phosphate-buffered saline) and lysed with 1 ml buffer 1 (10 mM Tris-Cl,pH7.5, 10 mM NaCl, 3 mM MgCl₂, 0.5% Nonidet P-40, 0.5 mMphenylmethylsulfonyl fluride [PMSF]). Cells were scraped into aneppendorf tube and put on ice for 10 min. The nuclei were collectedafter centrifugation at 5000 rpm for 10 min. Nuclear proteins wereprepared by resuspending the nuclei in buffer 2 (20 mM Hepes, pH7.9, 5mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 300 mM NaCl, 20% glycerol, 0.5 mMPMSF), after centrifugation at 14,000 rpm for 10 min the supernatant washarvested as nuclear extract. Double-stranded AP-1 oligonucleotide(Santa Cruz Biotech, Inc.) was end-labeled with [γ-³²P] ATP (DuPont NEN)using T4 polynucleotide kinase (Pharmacia). Labeled probe was separatedfrom free nucleotide through a Sephadex G-50 mini-spin column(Pharmacia). DNA-protein binding was performed by mixing 10 μg ofnuclear extract with ³²P-labeled double-stranded AP-1 consensusoligonucleotide in a total volume of 20 μl containing 20 mM Hepes, pH7.9, 1 mM MgCl₂, 4% Ficoll, 0.5 mM DTT, 50 mM KCl, 1 mM EDTA, 2 μgpoly(dI.dC) and 1 mg/ml BSA for 45 min on ice. The DNA protein complexwas separated on a 4% native polyacrylaminde gel using 0.5×Tris-borate-EDTA buffer at 150 V. Gels were then dried andautoradiographed.

Example 3 Overexpression of RHAMM Activates Expression of c-fos andc-jun, and Matrix Metalloproteinases Associated with Response-to-InjuryProcesses

Expression of the transcription factors c-fos, c-jun, jun B areassociated with response to injury processes in mammalian tissues.Northern analysis was used to show that of c-fos, c-jun, and jun B butnot jun D expression are stimulated by overexpression of the transitionstage hyaladherin, RHAMM.

Briefly, cells were grown in DMEM with 10% FBS and were starved in theabsence of serum for 6 h. Cells were washed twice with PBS and total RNAwas isolated by guanidine isothiocyanate method. In concise, cells werelysed in 4 ml solution D (5.3 M guanidine isothiocyanate, 30 mM sodiumcitrate, 0.7% N-laurylsarcosine, 0.72% 2-mercaptoethanol). To eachsample, added 4 ml of acid phenol, 1 ml of chloroform and 0.45 ml of 2 Msodium acetate. The solution was mixed well and centrifuged at 7000 rpmfor 30 min, the aqueous phase was collected and precipitated with anequal volume of 2-propanol. Pelleted RNA was dissolved in 50 μl diethylpyrocarbonate (DEPC) treated water. The RNA was second extracted with0.4 ml of TRIzol reagent (GibcoBRL) with the addition of 0.1 mlchloroform. After vigorous mixing and centrifugation, the RNAsupernatant was precipitated with 2-propanol and washed in 75% ethanol.Finally, the RNA was dissolved the DEPC-treated water. The expressionlevel of c-fos, c-jun, jun B and jun D were probed with a rat c-fos anda human c-jun probe, respectively. The blots were stripped and re-probedwith rat GAPDH cDNA as internal standard. The results, as shown in FIG.7, show that expression of c-fos, c-jun, jun B but not jun D isstimulated in LR21 cells that overexpress RHAMM. In addition, LR21 cellswhich overexpress RHAMM constitutively form podosomes and form few focaladhesions (data not shown).

C-fos and c-jun expression are stimulated in LR21 cells in comparison toparental 10T1/2 whether or not they are grown on fibronectin (FIG. 8).Briefly, cells were grown in DMEM with 10% FBS and cell monolayers weretrypsinized. Cells were washed and plated on fibronectin andpoly-1-Lysine coated dishes in the medium without serum. These tissueculture dishes were precoated with 25 μg/ml fibronectin or 100 μg/mlpoly-l-Lysine at 4° C. for overnight and washed twice with PBS beforeuse. Cells were then incubated under this starving condition for 6 hr.total RNAs were extracted and hybridized as previously described. Thelevels of c-fos and c-jun were determined by hybridization with a ratc-fos cDNA and a human c-jun cDNA. The blot was stripped and re-probedwith rat GAPDH cDNA as internal control. These results, as shown in FIG.8, further illustrate that a cell culture overexpressing a transitionmolecules such as RHAMM exhibits an activated signaling phenotypecharacteristics of transition stage cells.

Another characteristic of transition cells is an increase in theexpression of matrix metalloproteinases. This increase inmetalloproteinases expression is exhibited in cells that overexpressRHAMM and these cells show reduced expression of matrixmetalloproteinase inhibitors. The association of increased RHAMM andmetalloproteinases activity is demonstrated by Northern analysis ofRHAMM and matrix metalloproteinase mRNA levels in 102T1/2 and LR21 celllines as illustrated in FIG. 9.

Briefly, cells were grown in DMEM with 10% FBS and were starved for 6hours in the medium without serum. Cells were washed twice with PBS andtotal RNA was isolated by guanidine isothiocyanate method. In concise,cells were lysed in 4 ml solution D (5.3 M guanidine isothiocyanate, 30mM sodium citrate. 0.7%0 N-laurylsarcosine. 0.72% 2-mercaptoethanol). Toeach sample, added 4 ml of acid phenol, 1 ml of chloroform and 0.45 mlof 2 M sodium acetate. The solution was mixed well and centrifuged at7000 rpm for 30 min, the aqueous phase was collected and precipitatedwith an equal volume of 2-propanol. Pelleted RNA was dissolved in 50 illdiethyl pyrocarbonate (DEPC) treated water. The RNA was second extractedwith 0.4 ml of TRIzol reagent (GibcoBRL) with the addition of 0.1 mlchloroform. After vigorous mixing and centrifugation, the RNAsupernatant was precipitated with 2-propanol and washed in 75% ethanol.Finally, the RNA was dissolved the DEPC-treated water.

Denatured RNA samples of 20 μg were separated in 1% agarose gelcontaining 2.2 M formaldehyde, transferred to a Zeta probe membrane(BioRad), cross-linked with an ultraviolet cross-linker (Strategene).The membrane was prehybridized in 0.35 M phosphate buffer containing 1%BSA, 7% SDS and 30% formamide for 5-6 h at 55° C. The expression levelof RHAMM, gelatinase B, and stromelysin were detected by hybridizing themembrane with a ³²P-labeled cDNA of a mouse full length RHAMMv2. Afterwashing the membrane in 0.5×SSC and 0.5% SDS at 55° C. for 1.5 h, themembrane was autoradiographed. The blot was subsequent stripped andre-probed with a mouse gelatinase B cDNA, a human stromelysin cDNA, or arat GAPDH cDNA as internal standard.

In addition to showing increased expression of metalloproteinases, cellsthat overexpress RHAMM also show decreased expression of inhibitors ofmetalloproteinase such as timp-1. Northern analysis of tissue inhibitorof matrix metalloproteinase (timp-1) expression in LR21 cell shows areduced level in comparison to normal quiescent cells as illustrated inFIG. 9. LR21 cells which overexpress RHAMMv4 show decreased expressionof timp-1, which normally blocks activity of metalloproteinases.

Example 4 Overexpression of RHAMM Restricts the Extent to whichCytokines and Growth Factors Activate ERK Signaling Pathways

As Previously mentioned, the overexpression of RHAMM produces atransition cell phenotype that only partially activates erk signalingpathways. This is further illustrated in FIGS. 10 and 11 which show thatsignaling molecules ordinarily fully activated by growth factorinduction are restricted, or partially activated by overexpression ofRHAMM.

FIG. 10 shows a phosphoprotein activity analysis that directlyillustrates that cells that overexpress RHAMM have elevated erkactivation of MAP kinases but that this activation is restrictedrelative to the level of activity observed in normal cells induced by agrowth factor (PDGF). The Figure shows both phosphoylation of erkmolecules and erk2 dependent phosphorylation of MBP molecules.

Briefly, cells were grown in DMEM with 10% FBS, and cells were starvedfor 6 h in the medium without serum. Cells were then stimulated withPDGF (25 ng/ml) for 30 and 60 min. Cell monolayers were washed threetimes with PBS and total cellular extracts were prepared in a buffercontaining 25 mM Hepes, pH 7.7, 100 mM NaCl, 2 mM MgCl₂, 0.2 mM EDTA,0.5% Triton X-100, 0.5 mM DTT, 20 mM P-glycerophosphate, 0.1 mM sodiumorthovanadate, 0.5 μg/ml leupeptin, 100 μg/ml PMSF. Cellular lysates of100 μg total protein were incubated with anti-ERK2 antibody conjugatedagarose (ERK(C-14), Santa Cruz Biotech., Inc.), immuno-complexes werewashed twice with the above lysis buffer and twice by kinase buffer (20mM Hepes, pH 7.7, 10 mM MgCl₂, 2 mM MnCl₂, 2 mM DTT and 25 μM ATP). ERK2activity was determined by in vitro kinase assay using 2 μg substrateMBP and 1 μCi [γ-³²P] ATP in 20 μl of kinase buffer. After incubation at30° C. for 20 min, the reactions were terminated with Laemmli buffer,and proteins were separated by SDS-PAGE, gels were dried andautoradiography.

The amount of ERK2 and phosphorylated MAPK were detected from the totalextracts by western blot analysis and ECL chemiluminescence system.Lysates of 25 μg total protein were resolved by 10% SDS-PAGE andtransferred onto nitrocellulose membrane (BioBlot, Costar) usingTrans-Blot® Semi-Dry Electrophoretic Transfer Cell (BioRad) with atransfer-blotting buffer containing 20 mM Tris. 150 mM glycine, 0.01%SDS and 20% methanol. The filters were blocked for non-fat skim milk inTBS-T (0.01% Tris, pH 7.5, 150 mM NaCl and 0.1% Tween 20) at 4° C.overnight. The membranes were then probed with phospho-specificanti-p44/p42 MAP kinase antibody (New England BioLabs, Inc) byincubation at room temperature for 1.5 h. After washing three times withTBS-T for 30 min, blots were incubated with horseradish peroxidaseconjugated anti-rabbit antibodies (NEB) for 1 h. The filters were washedthree times for 30 min. and visualized on X-ray film withchemiluminescence detection method (NEB).

As shown in FIG. 10, the results of this analysis shows that LR21 cellsoverexpressing RHAMMv4 are restricted in the extent to whichproinflammator, cytokines/growth factors (e.g. PDGF) can activate erkkinase.

FIG. 11A shows a Northern analysis of IL-1 induction of c-fos expressionin 10T1/2 and LR21 cell lines. Cells were grown in DMEM with 10% FBS andstarved for 6 hours in the medium without serum. Cells were thenstimulated with IL-1 (20 ng/ml) for 30 min and 60 min. Total RNAs wereextracted and hybridized as described above. The level of c-fos wasmeasured by hybridization with a rat c-fos cDNA. The blot was strippedand re-probed with rat GAPDH cDNA as internal control. The results showthat expression of c-fos in response to IL-1 and TNF is restricted inLR21 cells.

FIG. 11B shows a Northern analysis of TNF-α induction of c-fosexpression in 10T1/2 and LR21 cell lines. Cells were grown in DMEM with10% FBS and starved for 6 h in the medium without serum. Cells were thenstimulated with TNF-α (30 ng/ml) for 30 min and 60 min. Total RNAs wereextracted and hybridized as described above. The level of c-fos wasmeasured by hybridization with a rat c-fos cDNA. Again it can be seenthat the expression of c-fos in response to a injury response growthfactor i.e., TNF-α, is restricted in LR21 cells.

Example 5 RHAMM Overexpression Prevents Focal Adhesion Formation andInduces Constitutive Podosome Production

A key feature of cells over-expressing RHAMM, LR21, prepared asdescribed above is that they do not form focal adhesions, FIG. 12A showsthat the parent cell line, 10T1/2, form very discreet focal adhesions,as demonstrated with anti-vinculin staining. In contrast LR21 cells donot form focal adhesions (FIG. 12B). This inhibition of focal adhesionformation may be responsible for the lack of response of these cells tocytokines such as IL-1 and TNF. It would appear that as long as cellsare expressing RHAMM they do not form focal adhesions and remainunresponsive to cytokines.

In addition, 10T1/2 cells, the parent cell line when plated form smallnumbers of podosomes immediately following plating as shown in FIG. 13.By 12 to 24 hours, there is little formation of podosomes and there isnow the formation of focal adhesions in these cells. In contrast to10T1/2 cells, LR-21 cells that over-express RHAMM form podosomesconstitutively. The level of podosome formation is higher and continuousin cells over-expressing RHAMM. These data indicate that RHAMMoverexpression is required for podosome formation in cells immediatelyfollowing injury or sustained disease conditions.

Example 6 Blockage of ERK Activity Inhibits the Formation of PodosomesAnd Migration of Cells Toward Wounds Promoted by Overexpression RHAMM

To further illustrate the relationship between RHAMM, erk activity andpodosome formation in transient stage cells, podosome formation is shownto be inhibited by inhibitors of erk activity. FIG. 14 shows thatenhanced podosome formation resulting from RHAMMv4 overexpression, isblocked by inhibitors of erk kinase which also blocks cell migrationinto wound sites. Overexpression of RHAMMv4 results in a sustained highproduction of podosomes, detected by the marker protein cortactin (A).Inhibition of erk kinase by PD09058 reduces the number of podosomes (B)as does mutation of intracellular RHAMMv4 (C) so that erk does not bindto RHAMM. Overexpression of RHAMMv4 enhances cell migration into wounds(D) compared to the parent 10T1/2 fibroblast line that produces littleRHAMMv4 (E). The addition of PD09058 blocks wound repair of RHAMMv4overexpressing cells (F).

Example 7 RHAMM is Transiently Detected on the Surface of Cells and isRequired for Podosome Formation and Cell Motility: Method of DetectingTransient Cells

Exon 3 and 4 of RHAMM provide peptides and antibodies thereto which areuseful for detecting RHAMM expression and demonstrating that RHAMM isassociated with podosomes. FIG. 15A shows a comparison of the expressionof RHAMM at the cell surface using anti-exon 4 RHAMM antibody orantibody “R3.8” raised against a whole RHAMM polypeptide. The chartsummarizes the results of FACS analysis of in vitro growth of invasiveMDA231 cells in comparison to MCF-7 human breast cancer cells which arenon invasive cells. The results show that cell surface RHAMM is presentin larger amounts on MDA-231 cells than on MCF-7 cells.

FIG. 15B shows the amino acid sequences of murine and human RHAMMpeptides including: a peptide from murine exon 3 (SEQ. ID NO: 14); asmaller peptide used to raise anti-exon 3 antibodies (SEQ. ID NO: 15); apeptide from murine exon 4 (SEQ. ID NO: 16); a smaller peptide used toraise anti-exon 4 antibodies (SEQ. ID NO: 17), a human RHAMM peptidefrom exon 5 (SEQ. ID NO: 18); a human RHAMM peptide homologous to murineexon 3 (SEQ. ID NO: 19); and a murine RHAMM peptide homologous to humanexon 5 (SEQ. ID NO: 20). The human exon 4 homologue is identical to themurine sequence used to raise anti-exon 4 antibodies. The C residuesshown in parenthesis were added during synthesis of the peptides.

FIG. 16 shows that cells treated by administering peptides mimickingexon 3, i.e., SEQ. ID NO: 15 (peptide 1, panel A) block the motility ofinvasive cells relative to a scrambled peptide (peptide 2, panel B).This effect is quantified in the graph shown in 16C and is highlysignificant (P<0.001, student's T test). FIG. 16D shores thatadministration of antibodies against peptides mimicking exon 4 (i.e.antibodies to SEQ. ID NO: 16) also inhibit the motility of invasivecells.

FIG. 17, panel A are micrographs that show that podosome formation isenhanced on the perimeter of LR21 cells that overexpress RHAMM incomparison to control 10T1/2 cells: Panel B shows that administration ofexon 4. i.e., peptide SEQ. ID NO: 16 (peptide 1) blocks the formation ofpodosomes while scrambled exon 4 peptide (peptide 2) does not. Podsomomeformation was visualized using either fluorescent cortactin or CAS, thelatter being a particularly useful marker for podosomes as illustratedby the micrographs in FIG. 17B.

In FIG. 18, MDA-231 cells were treated with hyaluronan together withanti-RHAMM antibody (exon 4 antibody). The antibody blocked theformation of podosomes as detected by cortactin staining.

FIG. 19 is a chart that quantitatively shows that anti-RHAMM antibodiesblock the rapid motility characteristic of MDA231 human breast cancercells but have only a small effect on the less rapid motility of cellsof the benign MCF human breast cancer cell line.

FIG. 20 further shows that anti-RHAMM antibodies inhibit the ability ofMDA231 cells to invade in vitro. The chart in 20A illustrates theinvasiveness of a variety of cell lines while 20B shows the ability of avariety of RHAMM antibodies to reduce invasiveness of MDA231 cells.

FIG. 21 shows that RHAMM binds to fibronectin but is blocked by antibodyto exon 3 indicating that exon 3 contains a fibronectin binding domain.FIG. 21 further illustrates that RHAMM binds to the CS-1 fragment offibornectin and not to the RGDS sequence which was previously consideredto be a critical sequence for fibronectin signaling of matrix proteindegradation. Panel A shows that RHAMM binds to fibronectin as detectedby an ELISA. Panel B shows that exon 3 of RHAMM binds to fibronectin butnot through the RGDS region but rather through the CS-1 region. Panel Cshows that peptides mimicking exon 3 are able to block the binding ofintact RHAMM to fibronectin, providing a rationale for why peptidesblock cell locomotion and podosome formation.

Example 8 ERK Kinase Involved in Cellular Motility

Elevated erk activity is associated with, and required for, rapid cellmotility characteristic of proliferative or invasive cells such as thebreast cancer cell line MDA231 which express high levels of RHAMM.

The relationship between erk activation and cell motility is illustratedin FIG. 22 which shows that when cells overexpress RHAMM such as in thecase of MDA23) cells, the level of cell motility is high. When erkkinase activity is inhibited by treatment with a MEK inhibitor(PD09058), that inhibition strongly reduces cellular locomotion andblocks cell invasion. FIG. 22 further shows that MDA231 cells expressinga mutant version of RHAMM (HA mutant) have reduced mobility. Therelationship between RHAMM expression and cell motility is furtherestablished by treating MDA231 cells with anti RHAMM antibodies,resulting in reduced cell mobility.

Example 9 Overexpression of RHAMM Promotes Podosome Formation

Podosomes are transient structures at lamellae tips that are requiredfor the efficient release of the MMPs. Together with other proteinases,MMPs initiate extracellular matrix remodeling. This initial remodelingof matrix attracts white cells to the site of injury, providingadditional source of pro-inflammatory cytokines and growth factors thatare responsible for the amplification of the response-to-injury. Thisexperiment shows that transient RHAMM overexpression will alter podosomeformation in transfected 10T1/2 cells.

Briefly, RHAMMv4 cDNA was tagged with HA and transfected into 10T1/2cells, 10T1/2 cells were cultured to 40-50% confluence and transfectedwith 10 μg of RHAMMv4 CDNA tagged with HA in 60 μl of superFect reagent.After five hours of incubation, monolayers were washed twice with PBSand the transfected cells ere cultured an additional 48 hours withgrowth medium supplemented with 10% FBS. The cells were harvested inRIPA buffer and RHAMM expression was detected by Western analysis. Onlythe transfectants that expressed similar level of (2-3 fold higher thanparenthal cells) were used for assays. HAv4tag cells were selected andused in this experiment. For the experimental purposes transfected cellswere plated on the fibronectin (FN) substrate at the 50% density. Tovisualize the presence of Hav4tag cDNA cells were stained at differenttime points: ½ h, 2 h, 6 h, and 24 h respectively with monoclonalantibody against HA. Additionally, cells were stained with themonoclonal Ab against HA (dil.1:50) 1 h at room temperature. Cells werethen washed with 1% BSA in PBS and incubated with x4 antibody. X4antibody was detected by Texas-red (1:100). Cells were incubated 1 h atroom temperature in Texas red. Some cells were plated onto RITC-labeledfibronectin in order to detect the ability of cells to digest thisextracellular matrix protein providing an assessment of the functionalcapability of the podosomes. A clearing of fluorescence indicates thatcells have released collagenases that are able to digest fibronectin.

In both experimental paradigms, results were examined under the confocalmicroscope.

As shown on FIG. 23A, at the 2 h point 100% of plated cells formedpodosomes. Additionally, v4 tagged RHAMM cDNA was found in perinuclearregion as well as in the podosomes. Evidence that podosomes made byRHAMM transfected cells are releasing proteases is provided in FIG. 23Bwhich shows the clearing of FITC-fibronectin underneath the platedcells. The dark area indicates that fibronectin has been proteolyzed andreleased from the cell substratum. Based upon this experiment it isevident that overexpression of RHAMM promotes podosome formation in10T1/2 cells.

Example 10 Antibodies Against TAM Domains and Leucine Zipper InhibitPodosome Formation

The objective of this experiment was to investigate whether RHAMMinduces podosome formation in a system where RHAMM surface sites wereblocked with a exon4 (TAM) A antibody. Leucine zipper peptide (LZP) wasalso tested for its capability to compete for RHAMM surface sites withfibronectin, since this is the binding site for fibronectin.

Briefly, LR21 cells were plated in DMEM with 10% serum at 70-80% densityand allow to grow for 8 h. Cells were then washed twice with PBS. Afterbeing washed cells were incubated in cell dissociation medium to detachfrom the plates. Cell dissociation medium was harvested and centrifugedat 1000 rpm's for 3½ min. Then, cells were plated at fibronectin-coatedcoverslips at 50% density in DMEM supplemented with 10% FBS. Cells wereallow to grow for up to 9 h. Plates were then divided into 4 groups andtreated in the following manner: control group was treated with 50 μg/mlof BSA in DMEM supplemented with 10% FBS; second group was treated with50 μg/ml of v4 antibody; third group was treated with 100 mg/ml of LZPand the fourth group was treated with combination of v4 antibody and LZPat the same concentrations as they were used in separate treatments.Cells were kept with the proteins for 30 min and they were fixed with 3%paraformaldehyde. Cells were stained with cortactin (dil.1:100) for 1 h.Subsequently, cells were washed with 1% BSA in PBS and stained withTexas-red mouse IgG (dil. 1:100). Staining of the cells was examined byconfocal microscope.

LR21 cells were plated onto fibronectin substrata as outlined above for8-12 hrs in serum free medium in the presence of IgG alone or anti-TAMantibody (“exon 4”). The supernatant culture medium was collected atthat time and concentrated on an amicon filter that retains proteins ofover 20 kDa. The retentate was suspended in loading buffer withoutmercaptoethanol or SDS-PAGE and run on a polyacrylamide gel impregnantedwith gelatin. The gel was then incubated in PBS containing Mg++ and Ca++buffer to permit collagenase activity at 37 C for several hours. The gelwas washed and stained with Coomassie Blue. Cleared areas indicate thatcollagenases released into the supernatant medium by LR21 cells isactive.

As shown on FIG. 24A, v4 antibody added to the medium competed for theRHAMM binding sites with fibronectin which resulted in reduced podosomeformation by those cells up to 25% compared to the BSA-treated control.LZP and combination of LZP and v4 antibody didn't result in any changesin podosome number. The reason for this result could be the fairly highconcentration of LZ peptide used in this experiment.

Based upon these results it is evident that RHAMM on the cell surface isrequired for the efficient podosome formation. Addition of v4 antibodiescompeted with fibronectin for the RHAMM binding sites which resulted inlower podosome formation by LR21 cells. The antibody also blockedrelease of collagenase (FIG. 24B), consistent with its blocking podosomeformation.

Example 11 RHAMM v4 and Full Length RHAMMv5 Interact With erk 1 Kinase

The most common murine RHAMM RNA transcript encodes a 95 kDa protein(referred to as “v5”). In addition, a shorter form of RHAMM may existencoding a 73 kDa protein (v4), which lacks 163 N-terminal amino acidsfound in the longer RHAMM form. The objective of this experiment was todetermine which form of RHAMM associates with erk and which particulardomain of RHAMM is responsible for this interaction.

A. In Vitro Binding Competition Assays.

Purified GST-RHAMM proteins were released from GST with trombin andRHAMM was coupled to Amino Link plus coupling gel (Pierce). Afterseveral washes with PBS, RHAMM-coupled beads were incubated withpurified erk1 His-6-tagged fusion proteins in binding buffer for 1 h at4° C. on Nutator rotor. After several washes with cold binding buffer,the beads were boiled for 2 min in cold loading buffer, then proteinswere separated on SDS-PAGE and transfer to nitrocellulose blots forwestern analysis. Anti-erk1 antibody (K23) was used to detect thiskinase on western blots. For competition assays, 1 μg of purified erk1His-6 fusion protein was incubated with 10 μg of soluble RHAMM proteinfor 1 h at room temperature, then incubated with beads-RHAMM for anadditional 1 h. For peptide competition assays, 1 μg of erk1 His-6fusion protein was incubated with beads-RHAMM for another 1 h on aRotator. Three different peptides were used in competition bindingassay, D4: QEKYNDTAQSLRDVTAQLESV (SEQ ID NO:50), D5:KQKIKHVVKLKDENSQLKSEVSKLRSQLVKRK (SEQ ID NO:51), and P-16 peptide:CSTMMSRSHKTRSHHV (SEQ ID NO:26).

B. Immunoprecipitation.

Parental 10T1/2 cells and transfected cell lines were plated at 50%confluence for 6-24 h and washed two times with cold PBS and lysed in alysis buffer, containing leupeptin (1 mg/ml), aprotinin (0.2 TIU/ml) anddichloroisocoumarin (200 μM). The lysates were centrifuged and equalamounts of protein (300-400 μg) from each sample were added to 2 μg ofanti-RHAMM antibody (R3.2), and anti-erk-1 (K23) antibody. After 1 h ofincubation at 4° C. on a Nutator rotor, 50 μg of a 50% solution ofprotein G Sepharose was added and incubated at 4° C. for an additional 1h. then washed four times with lysis buffer.

C. Western Analysis.

Cells were plated at 50% confluence and grown for 6-24 h. Then,monolayers were washed with cold PBS, lysed in RIPA buffer and subjectedto SDS-PAGE. Separated proteins were transferred onto nitrocellulosemembranes (BioRad) using a Transfer buffer. Non-specific binding siteswere blocked with 5% defatted milk in Tris buffer. RHAMMv4 antibody wasprepared against following sequence: VSIEKEKIDEK (SEQ ID NO:50). RHAMMv5antibody was prepared against following sequence: QERGTQDKRIQDME (SEQ IDNO:21). Membranes were washed three times with TBST, then incubated withhorseradish peroxidase-conjugated goat anti-rabbit IgG (1;10000) for 30min at room temperature. Bound antibody was visualized bychemiluminescence (ECL). The densitometry was performed with amulti-Analyst program (Bio-Rad). To determine antibody specificity,anti-RHAMM antibodies were incubated with beads-linked with RHAMMprotein (1 μg antibody/20 μl beads) for 1 h at 4° C. on a Rotator, andthen centrifuged for 5 min. The supernatant was used to probe membranes.

Results are shown in FIG. 25. Briefly, FIG. 25B shows erk1 binding to v4and v5 obtained in vitro, whereas FIG. 25C shows similar results invivo. The Bottom Western blot represents total cellular erk kinase anddensitometery was calculated as the ratio of total cellular erk versuserk associated with RHAMM. Both RHAMMv5 and v4 associated with erk1kinase. RHAMMv4 more strongly activates erk kinase than RHAMMv5 (FIG.25D), which contains all of the domains of v4 but in addition N-terminalsequence that negatively regulates the functions of the activating D2-5domains (FIG. 25B). The presence of mutations in both D5 or D4 domainsor in competition assays the presence of both D4 and D5 peptides,reduced erk1 binding to v4 by 90% (FIG. 25C), suggesting key roles ofthose domains in binding.

Thus, in summary both forms of RHAMM (v4 and v5) associate with erk1 invivo and in vitro but only the short form strongly activates the erkkinase cascade. The hyaluronan binding domains (See FIGS. 25A, D5) and arepeated sequence (D4) are required for binding of erk1 to RHAMM.However, both D3 (encoding the TAM domain) and D2 (encoding the leucinezipper) are required for activation of erk kinase although they are notinvolved in the binding of erk1 to RHAMM.

Example 12 HA Binding Domains of RHAMM Peptides and Antibodies Theretofor Affecting a Response-to-Injury Process

FIG. 26 illustrates that HA binding peptides including artificial mimicsof hyaluronan binding domains of RHAMM are able to block cell motilityin podosome forming cells while scrambled peptides do not. FIG. 26Aprovides the sequence of several artificial HA binding peptides of theformula BX7B (SEQ ID NO:28) discussed above. Panel B shows that each ofthese peptides are able to block cell motility when administered tocells. Panel C shows that an HA binding peptide according to one of thegeneral structures provided in SEQ ID NO: 1-5, and more particularly,having one of the structures provided in SEQ. ID NOS. 6-10 is even moreeffective in blocking cell motility and that a scrambled version of thispeptide is not.

Example 13 HA-Binding Peptide Mimetic (P-16) and RHAMM Sequence Peptide(423-43′ AA) Inhibit Migration of Human Fibroblast

Wound healing is the response to injury. By day three after thewounding, fibroblasts appear in the fibronectin-fibrin framework andinitiate collagen synthesis. Fibroblast proliferate in response togrowth factors present on the wound site and this complex series ofcellular and inflammatory processes resulting in deposition ofconnective tissues and its remodeling into the scar tissue. Thefibroproliferative response is accompanied with wound contraction andfibrosis due to the presence of myofibroblasts and to the enhancedproduction of collagen. In adult humans, the extracellular matrix isremodeled to sustain and direct the cellular changes and to restore thetissue integrity. Such exuberant healing responses often lead to tissuefibrosis and contraction commonly referred to us as scarring. Fibrosisof adult human tissue is a serious clinical problem that results inmalfunction of tissue due to, for example: formation of intraabdominaladhesions, cirrhosis of liver, failure of anastomoses as well asadhesions following injury.

In animal models of skin wounding, expression of an active (73 kDa)RHAMM form is transiently increased early after injury and this elevatedexpression occurs in most cell types present in the wound site. Aspecific domain within RHAMM (D5) that is responsible for interactionsof hyaluronan with cell surface RHAMM and erk1 binding to intracellularRHAMM was identified and utilized to develop a peptide mimetic reagent(p-16), which blocks function of cell surface RHAMM. Another RHAMMsequence consisted of 9 AA (423-432) which was also tested in thefollowing experiment.

The objectives of following experiment were to test the abilities of twoRHAMM synthetic peptides to inhibit migration of human fibroblasts. Oneexperimental model tested a 16 amino acid RHAMM peptide mimetic(P-peptide) to inhibit migration of Human Foreskin Fibroblast (HFF)through the wound gap. Another peptide consisted of 9 AAs (RHAMMsequence, 423-432 AA) was also tested in regards of cell locomotion ofhuman fibroblasts.

Experiment A.

Human fibroblasts were seeded at 5×10⁵ cells/well in 6 well plates usingα-MEM supplemented with glucose and 10% FBS. After being 6 hours in theculture (80-90% confluency), cells were injured with the single edgecell scraper (one injury/dish). Cells were washed twice with PBS andtreated with two different concentrations of P-peptide (10 μg and 100μg) for 15 h. Untreated cells served as control. Following 15 hours ofincubation, images were taken using a 5× modulation objective (Zeis,Germany) attached to the Zeiss Axiovert 100 inverted microscope equippedwith Hoffman Modulation contrast optical filters (Greenvale, N.Y.). Thenumber of migrated cells in each image was counted choosing the ˜70% ofthe middle of each injury. Statistically significant (P<0.05)differences between means were assessed by the unpaired Student's t-testmethod, using Microsoft Excel '97 software.

Experiment B.

To quantify the effect of RHAMM sequence (423-432 AA) to alter velocityof cell locomotion human fibroblasts were seeded on T-12.5 fibronectincoated flasks using α-MEM supplemented with glucose and 10% FBS. 2.5×10⁴cells were seeded and cells incubated for 4 hrs at 37° C. Afterincubation time cells were treated with increasing concentrations ofRHAMM sequence peptide (423-432 AA, 0.1, 1.0, 5.0, 10 and 50 ng/ml) andcell locomotion was monitored over the period of 16 hrs on a 37° C.using 10× modulation objective (Zeiss, Germany) attached to a ZeissAxiovert 100 inverted microscope equipped with Hoffman Modulationcontrast optical filters (Greenvale. NT). Cell images were captured witha CCD video camera module attached to a Hamamatsu CCD camera controller.Motility was assessed using Northern Exposure 2.9 image analysissoftware (Empix Imaging, Mississauga, Ontario). Nuclear displacement of7-10 cells was measured and data were subjected to statistical analysis.Statistically significant (P<0.05) differences between means wereassessed by the unpaired Student's T-test method, performed usingMicrosoft Excel “97 software.

Results are shown in FIGS. 27 and 28. Briefly, FIG. 27 shows thattreatment of injured cells with 100 μg/ml of P-peptide inhibitedmigration of HFF cells approximately 4 fold compared to control cells(P<0.01). Lower concentration (10 μg/ml) of P-peptide didn't have anyeffect. As shown in FIG. 28, different concentrations of RHAMM sequence(423-432 AA) progressively inhibited migration of human fibroblasts upto 40%.

Both treatments were successful in inhibition of cell migration invitro. These important data suggests potential implementation of theboth P-peptide and RHAMM sequence 423-432 AA peptide in prevention oftissue contraction and fibrosis and ultimately prevention of abnormaltissue remodeling and scaring.

Example 14 Fibroblasts from RHAMM Knockout Mouse Produce Two Times LessMMP's Than Wild Type

MMP expression is involved in a wide variety of inflammatory diseasesand cancers. This experiment investigates whether fibroblasts which areobtained from RHAMM knockout mice have altered MMP production.

Briefly, embryonic stem cells (ES) were transfected with antisense cDNAthat recombined with the RHAMM gene, resulting in recombination and agenetic deletion of the RHAMM gene. The ES cells were injected intomouse blastocysts, and placed into pseudo-pregnant mice. Mice from theresultant litters were crossed and examined for the presence of agenetic deletion, in order to determine germ line transmission. Founderswere identified and homozygotes obtained.

Embryos from normal and knockout mice were taken out at the 13^(th) dayof their intrauterine development. Tissue was cut and tripsinized in theincubator at 37° C. for 10 min. Cell suspension was pipeted up and downseveral times in order to release fibroblasts from the tissue. Then,fibroblasts were plated on the Petri dishes (one embryo per one Petridish). Cells were grown for 2 days before first passage was done. Fivepassages were done before actual experiment was performed.

For experimental purposes, fibroblasts were plated on 6-well dishes,normal ones and fibronectin coated, both at the 70% confluency. Cellswere grown in DMEM medium for 2 h. After 2 h, medium was changed to DMEMwithout serum and cells kept in starvation medium for 24 h. Then, mediumwas taken out and the amount of gelatinase released into medium measuredby zymografic analysis. Briefly, DMEM medium taken out from plates wasrun on overnight in a cold room (+4° C.) on 10% acrylamide gelcontaining 1 mg/ml of gelatin. Then, gel was washed in TritonX-100 for 1h and subsequently incubated in a buffer on 37° C. for 24 h in order todevelop zymogram. Then, gel was stained with commassi blue whereas areaswith MMPs were left unstained. Intensities of unstained bands weremeasured and presented as relative numbers.

Results are shown in FIG. 29. Briefly, MMP release from knockoutfibroblasts is approximately 2.5 times lower compared to normal ones.

Thus, it is evident that RHAMM expression regulates production of MMPs.Absence of RHAMM in knockout fibroblasts resulted in marked decrease inMMP production.

Example 15 RHAMM Influences erk Phosphorylation upon PDGF Treatment inMouse Primary Fibroblasts

The purpose of this experiment was to investigate if erk phosphorylationis decreased in primary fibroblasts of the RHAMM knockout mouse.

Briefly, mouse normal and RHAMM knockout fibroblasts are plated in DMEMmedium and starved overnight. Medium was changed and two differentconcentrations of PDGF added. After 10 min cells were lysed in RIPAbuffer. Western blot analysis was done and proteins separated bySDS-PAGE. Bands were visualized by phospho-specific erk antibody.Subsequently, blot was stripped and reprobed with erk antibody.

Results are shown in FIG. 30. Briefly, erk phosphorylation is influencedby RHAMM expression: knockout mice exhibited at least two folds lowerphosphorylation of erk1 isoform compared to wild type. PDGFconcentration of 1 ng/ml produced the largest decrease (2.3 fold) in erkphosphorylation (FIG. 30). Thus, deficiency in RHAMM expression inknockout fibroblasts down regulates the capability of PDGF to activateerk pathway

Example 16 Fibroblasts Isolated from RHAMM Knockout Mouse Locomote atSignificantly Lower Rate than Wild Type Fibroblasts

This experiment investigates the impact of lacking RHAMM expression oncell migration in mouse knockout fibroblasts.

Briefly, knockout and wild type mouse fibroblasts were plated in 100 mmPetry dishes (normal or gelatin and fibronectin coated) and grown innormal DMEM medium. Cells were plated sparsely and left for 2 h toattach, spread and start to migrate. Two hours after plating, cells werechecked for migration and pictures were taken from the same spot every15 min. The images were overlaid and cell migration analyzed bymeasurement of the migration distance.

Results are shown in FIG. 31. Briefly, knockout fibroblasts havedecreased cell motility compared to wild type by two folds. Combinationof gelatin and fibronectin coating seem to potentiate slower migrationof mouse RHAMM knockout fibroblasts. Thus, it is evident that mouseRHAMM knockout fibroblasts migrate at the slower rate compared to wildtype cells. Attenuation of cell migration is between 2 to 4 fold.

Example 17 Administration of RHAMM Peptides or Antibodies InhibitResponse-to-Injury Processes Associated with Macrophages in Injured LungTissue

Clinical diseases characterized by lung inflammation include emphysema,asthma, cystic fibrosis, new-born lung disease involving chronicrespiratory distress syndrome, and the acute respiratory distresssyndrome that affects accident victims. Local inflammatory responsesthat recruit macrophages into the lung result in destruction of alveolartype II cells, which make the surfactant responsible for normal lunginflation. The infiltration of macrophages and abnormal local tissueresponses result in further tissue destruction and disease. Thispathological sequence results in improper lung expansion. A commonresponse to injury in mammalian tissue is increased motility ofmacrophages and macrophage accumulation near a wound site.

As described in more detail below, the RHAMM peptide mimetics preventthese pathological events, particularly recruitment and activation ofmacrophages, from occurring following bleomycin-induced lung fibrosis(FIG. 32). In particular, the infiltration of inflammatory cells andlocal responses such as fibrosis is completely absent with treatment.Yet, no toxicity was observed in the animals even at very highconcentrations. Therefore, these reagents should be effective fortreating lung diseases that involve recruiting macrophages andinflammatory cells, as well as fibrosis.

This experiment shows that macrophage responses are inhibited byadministration of antisera to RHAMM peptides. More specifically. FIG. 33illustrates that a significant increase occurs in the motility ofmacrophages from both bleomycin and saline-treated animals at four daysafter intratracheal instillation (*p<0.01 versus control, p<0.01 versussaline and control). Normal rabbit IgG had no effect on macrophagemotility, but anti-RHAMM peptide aa antiserum inhibited macrophagemotility from both saline−(# p<0.01 versus saline) and bleomycin-treated(p<0.01 versus bleomycin, saline and bleomycin+normal IgG) animals tolevels observed in macrophages from untreated healthy control animals.Values represent mean and standard errors of five animals studied foreach condition with mean velocities calculated.

FIG. 34 illustrates motility of BAL cells four days after injury inresponse to administration of RHAMM peptides. Macrophages frombleomycin-treated animals showed increased motilities as compared tothose from control and saline animals (*p<0.001). Animals pretreatedwith Scrambled Peptide A showed the same motility as macrophagesobtained from animals injured with bleomycin. However, macrophages fromanimals treated with Peptide A prior to bleomycin-induced injury showedsignificantly lower cell locomotion than either bleomycin-injured orscrambled peptide treated controls (# p<0.001). Values represent meanand standard error with three animals studied for each condition and atleast 20 cells tracked per animal studied.

Based on these findings, peptide mimetic and antibody formulations canbe utilized in the treatment of a variety of “response to injury”indications, including for example, emphysema, asthma and the chronicrespiratory distress syndrome associated with newborn lung disease.

Example 18 Administration of HA Binding Peptides InhibitResponse-to-Injury Processes Associated with Fibrosis in Injured LungTissue

Increased N-acetyl-β-glucosaminidase activity is a known marker forfibrosis in lung tissue. FIG. 35A shows in vivo effects of a HA-binding(Peptide A) on N-acetyl-β-glucosaminidase activity of BAL cells obtained7 days after injury.

Briefly, bleomycin injury results in an increased glucosaminidaseactivity (* p<0.01 versus controls and saline animals). ScrambledPeptide A had no effect on the glucosaminidase activity whereas PeptideA significantly decreased glucosaminidase activity (# p<0.05 versusbleomycin alone and bleomycin+scrambled peptide A). Values representmean and standard error with five animals studied for each condition.

FIG. 35B illustrates that mRNA of collagen type 1a in lungs harvested 4days after injury is reduced in response to administration of HA bindingPeptide A. Collagen type 1a is common indicator of fibrosis in lungtissue injury models as used throughout this invention. An increase incollagen type 1a mRNA was observed by 4 days after injury in controltissue, however, this increase was completely inhibited byadministration of HA binding Peptide A, whereas scrambled Peptide A hadno effect on the mRNA expression levels for this collagen. The datashown are representative of three independent experiments.

The ability of HA binding peptides to inhibit fibrosis is furtherillustrated by histological analysis. FIG. 36 are micrographs from ahistological analysis of lung tissue treated with and without HA bindingPeptide A after bleomycin injury. Panels (a-c) show tissue aftertreatment with saline alone, and panels (d-f) show tissues injured bybleomycin treatment. Panel (d) shows a fibrotic morphology in thepresence of bleomycin alone while panel (e) shows the morphology in thepresence of bleomycin and scrambled RHAMM peptide. In contrast, panel(f) shows that injection of bleomycin-treated animals with the senseRHAMM HA binding Peptide A results in a normal lung architecture despitethe injury caused by bleomycin.

Example 19

Expression of RHAMM in Different Cells Present in Synovial FluidsIsolated from RA Patients

This experiment determines which cell type from the synovial fluids ofRA patients express RHAMM isoforms.

Briefly, samples of synovial fluids from different RA patients werecentrifuged at 1600 rpm's for 10 min and pellets resuspended in 2-5 mlof Blocking buffer (BB, 1% human serum albumin in HBSS). After counting,10⁶ to 2.5×10⁶ cells per ml. were taken into each tube. Cells werewashed once with 1 ml of BB and the pellets resuspended in 100 μl of BB.First antibody was added (dil. 1:100) and samples incubated for 30 minon ice. Along with the first antibody 20 μl of specific markers forcertain cell type present in the synovial fluid were added, as well.Rabbit IgG was used as a control. Samples were washed twice with 1 ml ofBB. After washing, secondary antibody was added (FITC, dil. 5:100) andcells kept 30 min on ice. Again, samples were washed twice, each timewith 1 ml of BB and fixed with 0.3 ml of 0.5% paraformaldehyde.Immunofluorescence was determined by flowcytometre.

Results, are shown on FIG. 37. Briefly, the majority of cells present insynovial fluid are neutrophils. Macrophage/monocyte cells are present as5-10% of cells and T cells are also present as a minority.Macrophage/monocyte cells exhibited the highest RHAMM expression. Insome cases the number of exon-4-positive cells was as high as 99.8%. Asimilar pattern was observed in neutrophil populations but thepercentage of positively labeled cells was between 54.6% and 99.3%. Tcells also express RHAMM isoforms, although to a lesser extent comparedto the other two cell types.

In summary, all tested RA patients expressed RHAMM on the surface ofcells present in synovial fluid. The most abundant cell type isneutrophils. In all tested patients more than 50% of neutrophil cellpopulation was X4-positive. Significant number of macrophages expressingx4 was uncovered: in all tested RA patients more than 75% ofmacrophage/monocyte population was labeled x4 positive.

Example 20 RHAMMx4 and RHAMM R3.8 are Present in the Synovium TissueSections From RA Patients

Rheumatoid arthritis is the most prevalent type of inflammatoryarthritis, affecting 1.5% of the human population. RA is characterizedby synovial hyperplasia, destruction of articular cartilage and bone andmacrophage infiltration into synovial joints. Cytokines like IL-1 arepresent in increased levels and they play a major role in production ofMMPs, such as collagenase and gelatinase.

In order to investigate if there is any RHAMM expressed in the synoviumtissue of RA patients, immunohistochemistry was done. Briefly, pannusformed from synovium tissue was isolated and embedded in wax. Threemicrons tissue sections were obtained and slides were heated on 58° C.for 30 min. To deparafinized slides the following procedure was done:tissue sections were washed in xylene three times each four minutes.After washing in hylene, slides were washed in 100% ethanol two timeseach three minutes. Additionally sections were washed in 96% ethanol thesame amount of time. Slides were then incubated in dH₂O two times eachthree minutes and once in PBS. Tissue on the slides was then marked withbarrier-pen. The activity of endogenous peroxidase was blocked with 0.3%of hydrogen peroxide for 10 min. Slides were washed with dH2O two timeseach 3 minutes and with PBS two times each 5 minutes. Unspecific bindingwas blocked with 1% bovine serum albumin (BSA) in PBS at 37° C. for 30minutes. Different dilutions of RHAMMv4 and RHAMM R3.8 antibodies weremade: 1:100, 1:50, 1:25) in 1% BSA-PBS and incubated with tissue samplesovernight at +4° C. Two tissue sections served as controls and they wereincubated with either rabbit IgG (at the same dilution as theantibodies) or with vehicle which was 1% BSA PBS, without primaryantibody. After incubation with primary antibodies, slides were washedwith PBS three times, 10 minutes each. Consequently, biotinylatedantirabbit IgG was added and slides kept at room temperature for 1 hour(dil.1:200 in BSA-PBS). Slides were again washed with PBS three timeseach 10 minutes. Additionally, Avidin-biotin ABC reagent was premixedand incubated with slides at room temperature for one hour. Slides werewashed with PBS three times each time 5 minutes. After washing. DABsolution was premixed and incubated with slides for 5 minutes at roomtemperature. Samples were washed with dH₂O three times each time 5minutes and counterstained with hematoxylene for 1-2 minutes. Sampleswere washed with regular water and dehydrated. For dehydration similarprocedure was done as for deparafinization only this time steps weredone backwards. Slides were mounted and left to dry overnight.

Results are shown in FIG. 38. Briefly, synovium tissue isolated fromjoints of RA patient was positively stained (brawn staining) with RHAMMexon4 (pictures A and B) and RHAMM R3.8 (pictures C and D). Areas ofsynovial lining cells are enriched in RHAMM staining which are mostlikely macrophage cell type, although other cell types in the RAsynovium also express RHAMM (pictures A, B. C and D). Controls BSA(picture E) and rabbit IgG (picture F) are unstained.

Hence, it is evident that RHAMM is present in high levels in humanarthritic joints.

Example 21 RHAMM Peptide Mimetic Inhibits Progression in ExistingMultiple Sclerosis (MS) Model

Multiple sclerosis (MS) is a major human neurological disease in NorthAmerica and Western Europe. Although the mechanism by whichdemyelination takes place in MS is not fully understood, it appears thatthe persistence of high levels of improperly assembled myelin which isprone to destruction is a leading cause for on set of the disease.Creation of ND 4 model of transgenic mice (Mastronardi et. al. J.Neurosci Res (93) Vol. 36 pp. 315-324) provides useful tool forinvestigation of the possible mechanism involving destabilization of themyelin membranes and appearance of distinctive features of MS disease.

The purpose of this experiment was to attenuate clinical signs ofdemyelination in MS by inhibition of function of the cells involved inpathological processes.

Briefly, transgenic mice (ND 4) bearing 70 copies of the transgene forDM20, a myelin proteolipid protein, were utilized for assignment ofscores based upon clinical signs of demyelination. Clinical signs whichwere assessed included general shaking, seizures, head jerk, hind-limband tail shiver, unsteadiness, wobbly gait and limp tail. Within eachsign score between 0-4 was given: where zero score means absent andscore 4 means constant and uncontrollable appearance of the sign of thedisease. Experimental groups of mice were divided into 4; each groupcontained 5 animals: one normal, one ND 4 mouse untreated and three ND 4mice treated with RHAMM mimetic-P-peptide. Animals were treated threetimes per week with 10 mg/kg of P-peptide intraperitonealy. Peptide wasresuspended in 300 μl of PBS.

Results are shown in FIG. 39. Briefly, treatment of ND 4 mice withP-peptide showed significant attenuation of clinical signs of MSsymptoms from 3 to 6 months of age (FIG. 39). Applied in a fairly highdosage (10 mg/kg), the peptide exhibited 2 fold inhibition of diseasesymptoms, without observing toxicological or lethal effects on animals.

Example 22 Scar Reduction: P-Peptide Reduces Collagen I and IIIExpression in Excisional Model of Rat Skin

Wound-healing responses to injury involve a complex series of cellularand inflammatory processes resulting in deposition of connective tissuesand its remodeling into the scar tissue. The fibroproliferative responseis accompanied by wound contraction and fibrosis due to the presence ofmyofibroblasts and to the enhanced production of collagen. In adulthumans, the extracellular matrix is remodeled to sustain and direct thecellular changes and to restore tissue integrity. Such exuberant healingresponses often lead to tissue fibrosis and contraction, commonlyreferred to as scarring. Fibrosis of adult human tissue is a seriousclinical problem that results in malfunction of tissue due tointraabdominal adhesions, cirrhosis of liver, failure of anastomoses aswell as adhesions following surgery.

A fibrotic wound response contrasts with repair of fetal skin woundwounds which exhibit reduced leukocyte infiltration, reduced fibroplasiaand altered extracellular matrix remodeling resulting in a non-scaredappearance of the healed wound. Additionally, hyaluronan accumulation issustained in fetal skin while its accumulation is only transient inwounded adult skin.

This experiment tests the ability of 16 amino acid RHAMM peptide mimetic(P-peptide) to reduce tissue fibrosis in a rat punch biopsy model ofskin repair.

A. Animal Model.

Three-month old female Sprague-Dawley (200-250 g) rats were anesthetizedwith Somnitol (1 ml/kg) and subjected to 4 mm full-thickness dorsalpunch biopsies. Series of the P-peptide concentrations (1 ng-20 mg) weremixed into a diluted bovine/1% collagen (type 1) suspension and appliedonce only per biopsy punch at the time of wounding. A 50 μl of thepeptide/collagen solution was applied to the punch biopsy wound andallowed to polymerize over several hours. Collagen was used as vehicleto stimulate inflammation and fibrosis as rat skin normally showsminimal fibrosis. Collagen alone (control wounds) does not influence therate of healing when compared to phosphate buffer saline. A twenty fourhours after dorsal punch biopsies, animals were anesthetized withIsofluorane inhalant with oxygen and nitrous oxide and the experimentaland control wounds (collagen alone) were excised. Samples were flashfrozen in liquid nitrogen for RNA extraction.

B. RNA Extraction.

Frozen wounds were homogenized in 1 ml of Trizol (Gibco, BRL) untilcompletely homogenous. After being homogenized, samples were incubatedat room temperature for 5 min and 200 μl of chloroform was added. Tubeswere tightly capped and shaken vigorously for 15 sec. Then, samples wereincubated at room temperature for 2-3 min. After incubation, sampleswere centrifuged at 11200 rpm's for 15 min. at 2-8° C. Upper aqueousphase was transferred to another tube, carefully not to disturbinterphase or organic phase of extract solution. After transfer, 10 μgof T-RNA was added into the tube along with 0.5 ml of isopropyl alcohol.Samples were incubated at room temperature for 10 min. Subsequently,they were centrifuged in picofuge at 11200 rpm's for 10 ml at 4-8° C.After being centrifuged, supernatant was removed and pellet washed oncein 751° ethanol. Samples were vortexed for 15 sec. and spun in picofugefor 5 minutes on 8800 rpm's at 4-8° C. Remaining ethanol solution wascarefully removed and RNA pellet allowed to air dry. Pellet wasdissolved in DEPC H₂O (Diethylpyrocarbonate). Concentration of RNA wasdetermined by spectrophotometer. RNA was aliquoted into 20 μg portionsand stored in −70° C. freezer until required.

C. RT-PCR Analysis.

Frozen wound samples (50-100 mg tissue) were homogenized in 1 ml ofTrizol reagent and RNA was isolated according to standard Trizol ReagentProtocol. For the synthesis of oligo-dT-primed cDNA. 2 μg of total RNA.1 μg of oligo(dT) primers and Moloney Murine Leukemia Virus ReverseTranscriptase (Gibco Brl # 2802′-013) were used. Following 1 hincubation at 37° C. the reaction was stopped by heating samples at 95°C. for 5 min and 2 μl of RT reaction mixture was used for PCR. PCRamplification was performed with platinum Taq DNA polymerase (Gibco BRL#10966-018) and specific primers for collagen I and III were used: 5,CGA TGT CGC TAT CCA GCT GA (SEQ ID NO:52) for collagen I and thefollowing primer 5 ATC AGT CAG CCA TCT ACC ACC (SEQ ID NO:53) was usedfor collagen type Ill. Thermal cycles for collagen type I and III wereas follows: denaturation at 94° C., annealing at 60° C. andpolymerization at 72° C. for 20 cycles. In addition, a set of primers ofa common housekeeping gene B-actin, were run in parallel on 1.5% agarosegel as a loading standard.

Results are shown in FIG. 40. Briefly, collagen production, which is amarker for fibrosis, was assessed by semiquantitative RT-PCR analysis ofcollagen type I and III mRNA within the wound site. Levels of collagentype I and III mRNA following P-peptide (1 ng/ml-20 mg/ml) applicationare shown in FIG. 40. Treatment of wound sites with P-peptide reducedlevels of collagen type I and III measured at 24 h post wounding.

Example 23 Scar Reduction: ED-1 Expression is Reduced by P-PeptideTreatment in Excisional Model of Rat Skin

Fibrosis of adult human tissues is a serious clinical problem thatresults in malfunction of tissue due to keloids, hypertrophic scars,anatomonic strictures, intraabdominal adhesions, cirrhosis of the liver,neurologic deficits following injury to the spinal cord, valvular heartdisease, burned-injured joints as well as failure of anastomoses andadhesions following surgery.

The P-peptide was assessed for its effect on the course of wound repairby measuring macrophage infiltration into the wound through themeasurement of ED-1 expression, a marker for macrophages andfibroblasts.

A. Animal Model.

Three-month old female Sprague-Dawley (200-250 g) rats were anesthetizedwith Somnitol (1 ml/kg) and subjected to 4 mm full-thickness dorsalpunch biopsies. Series of the P-peptide concentrations (1 ng-20 mg) weremixed into a diluted bovine/1% collagen (type I) suspension and appliedonce only per biopsy punch at the time of wounding. A 50 μl of thepeptide/collagen solution was applied to the punch biopsy wound andallowed to polymerize over several hours. Collagen was used as vehicleto stimulate inflammation and fibrosis as rat skin normally showsminimal fibrosis. Seven days after dorsal punch biopsies, animals wereanesthetized with Isofluorane inhalant with oxygen and nitrous oxide andthe experimental and control wounds (collagen alone) were excised.Samples were flash frozen in liquid nitrogen for RNA extraction.

B. RNA Extraction.

Frozen wounds were homogenized in 1 ml of Trizol (Gibco. BRL) untilcompletely homogenous. After homogenization, samples were incubated atroom temperature for 5 min and 200 μl of chloroform was added. Tubeswere tightly capped and shaken vigorously for 15 seconds. Then, sampleswere incubated at room temperature for 2-3 min. After incubation,samples were centrifuged at 11200 rpm's for 15 min. at 2-8° C. Upperaqueous phase was transferred to another RNAse free tube, carefully notdisturbing interphase or organic phase of extract solution. Aftertransfer, 10 Hg of T-RNA was added into the tube together with 0.5 ml ofisopropyl alcohol. Samples were incubated at room temperature for 10min. Subsequently, they were centrifuged in picofuge at 11200 rpm's for10 min at 4-8° C. After being centrifuged, supernatant was removed andpellet washed once in 75% ethanol. Samples were vortexed for 15 sec andspun in picofuge for 5 minutes on 8800 rpm's between 4-8° C. Remainingethanol solution was carefully removed and RNA pellet allowed to airdry. Pellet was dissolved in DEPCH₂O). Concentration of RNA wasdetermined by spectrophotometer. RNA was aliquoted into 20 μg portionsand stored in −70° C. freezer until required.

C. RT-PCR Analysis.

Frozen wound samples (50-100 mg tissue) were homogenized in 1 ml ofTrizol reagent and RNA was isolated. For the synthesis ofoligo-dT-primed cDNA. 2 μg of total RNA. 1 μg of oligo(dT) primers andMoloney Murine Leukemia Virus Reverse Transcriptase (Gibco Brl #28025-013) were used. Following 1 h incubation at 37° C., the reactionwas stopped by heating samples at 95° C. for 5 min and 2 μl of RTreaction mixture was used for PCR. PCR amplification was performed withplatinum Taq DNA polymerase (Gibco BRL #10966-018) and specific primersthat used for ED-1 is: for ED-1-5′ CGA TGG CAG GAC AGT AGT CGC (SEQ IDNO:54) and/or 5 AAG GCT GCT GTT GAA AGG ACG (SEQ ID NO:55).

Thermal cycles for ED-1 was as follows: denaturation at 94° C. annealingat 59° C. and polymerization at 72° C. for 28 and 29 cycles. Inaddition, a set of primers of a common housekeeping gene B-actin, wererun in parallel on 1.5% agarose gel as a loading standard.

Results are shown in FIG. 41. Briefly. RT-PCR analysis of mRNA isolatedfrom the wound site treated with P-peptide (1 ng/ml to 100 ug/ml) showeddown regulation of ED-1 expression at 7 days after injury in comparisonto the untreated wounds.

Thus, P-peptide reduces infiltration of macrophages into the site of thewound.

Example 24 RHAMM HA Binding Peptides Inhibit Macrophage InfiltrationFollowing Skin Wounding

Several key processes are involved in excisional wounding healing andscarring. These include local inflammation and infiltration ofmacrophages and neutrophils. The objective of this study was todetermine whether different HA binding peptides inhibit macrophageinfiltration.

The excisional wound healing rat model used and the method of localapplication of peptides was similar to that described in example 23.Tissue biopsies were removed and assayed for Glucosiminidase activity, abiological marker for macrophages.

As shown in FIG. 42, the data demonstrate that HA-binding peptides A(RGGGRGRRR; SEQ ID NO:27), B (RGGGRGGRR: SEQ ID NO:56). C (RGGGRGGGR;SEQ ID NO:57) inhibited the infiltration of macrophages in woundedbiopsies, whereas peptide D (RGGGGGGGR; SEQ ID NO:58) which has asimilar sequence but does not bind HA does not inhibit macrophageinfiltration in wounding. In addition the scrambled peptide A did nothave any effect on macrophage levels.

In conclusion these data demonstrate that HA-binding peptides inhibitmacrophage motility and infiltration in wounding, and thus havepotential to promote wound healing and reduce scarring.

Example 25 RHAMM Regulates Prostate Cancer Progression

This experiment investigates whether functional expression of the HAreceptor RHAMM is required for enhancement of CaP cell motility andinvasion in vitro.

Briefly, Dunning CaP cell lines (AT-1, MatLyLu) were grown in DMEMmedium supplemented with 10% FBS at 37° C. in a humidified atmospherecontaining 5% CO2. All cell lines were passaged every 3-4 days uponreaching confluency.

A. Immunofluorescence.

Cells were seeded sparsely on glass coverslip and incubated in growthmedia for 24 h. cells were then fixed with 3% paraformaldehyde andpermeabilized with 0.2% triton X-100. RHAMM was visualized by indirectimmunofluorescence using a polyclonal antibody to the C-terminus (Zram2.3, 1:100) and Texas red conjugated donkey anti-rabbit antibody(1:100). Images are obtained using a Zeiss laser scanning confocalmicroscope.

B. Western Blotting.

Cells were also grown to 50-60% confluency were lysed using RIPA buffer.Equal amounts of total cell protein were loaded onto a 10% SDS-PAGE gel.RHAMM was probed using a polyclonal antibody to the C-terminus (Zram,1:1000) and HRP-conjugated goat anti-rabbit antibody (1:5000). RHAMM wasvisualized by chemiluminescence.

C. Cell Motility.

Cell were seeded sparsely and grown in 25 cm² flasks overnight.Serum-free medium was used for the experiments. Random cell motility ofcells untreated, or treated with either RHAMM polyclonal antibody (Re4)or peptide mimicking the HA-binding domain over two hours was visualizedby videomicroscopy. Cell motility tracks were analyzed using a NorthernExposure software. Statistical analysis was performed on 100 cells perfield and statistical significance was determined using unpaired Studentt-test.

D. Cell Invasion.

Cell were grown to confluency in grouch media, detached, and equalnumber of cells were seeded in 24-well Matrigel invasion chambers. Cellswere left untreated with RHAMM peptide and allowed to invade for 24 h.For statistical analysis, 5 high-power fields(400×) were counted for thenumber of cells that invaded throughout the membrane. Statisticalsignificance was determined using unpaired Student t-test.

E. MMP Activity.

Cells were grown to confluency in growth media, detached and equalnumber of cells were seeded in 6-well plates uncoated or coated with 50%Matrigel in media. Cells were allowed to adhere for 1 h to thesubstrate, and then treated with the peptide mimicking the HA-bindingdomain of RHAMM (100 μg/ml) for 24 h in serum-free media. The activityof MMP secreted into the media was determined by zymography using 8%SDS-PAGE.

Results are shown in FIG. 43. Briefly, FIG. 43A shows the Western blotanalysis using a RHAMM polyclonal antibody detecting progressivelyincreasing expression of 54 kDa RHAMM isoform in proportion tomotility/invasivity: the highly motile/invasive subline. Fb2>the weaklymotile/invasive parental line. MC2>the nontumotigenic parental NbEepithelial line. FIG. 43B shows RHAMM localization to a sites of cellextension and to podosomes of invasive CaP cells. Open arrows point tosites of cell protrusion, whereas closed arrows point to circularstructures known as podosomes or invadopodia. Left panel FIG. 44A showsthat RHAMM regulates Dunning CaP cell line motility and invasion,whereas right panel of FIG. 44A showed that MatLyLu cells treated with aRHAMM peptide showed a significant reduction (p<0.025) of about 20% ininvasive potential as determined using Matrigel in vitro invasionchambers. However no effect of peptide was observed upon treatment ofthe AT-1 cells. FIG. 44B shows that secretion of MMP was higher in AT-1cells compared to MatLyLu cells when grown on plastic. Matrigel did notreduce MMP activity in AT-1 in MatLyLu. When RHAMM blocking peptide wasadded, MMP activity was suppressed.

Thus, RHAMM is preferentially expressed in more motile invasive andmetastatic CaP cells. Blocking RHAMM function significantly andpreferentially reduces motility, invasion, and MMP activity in highlymetastatic CaP cells.

Example 26 Influence of RHAMM Peptide Mimetic On Weight Gain in MurineModel of SLE

F1 (NZB/W) mice, hybrids of New Zealand Black (NZB) and Nest ZealandWhite (NZW) mice, are a murine model of SLE (Systemic LupusErythematosus). These mice develop spontaneously autoantibodies to DNAand other cell components. Female mice develop a more rapid diseasecourse than males, with death from renal failure occurring by 8-10months of age in females and 18-20 months of age in males. Females of 8weeks of age are free of overt symptoms of disease, with gradualdevelopment of autoantibodies, glomerulonephritis, proteinuria, renalfailure and death. The renal disease is likely secondary to the immunedysfunction.

In addition to progressive renal inflammatory disease, these mice shoreincrease in body weight of 20%-30%, which is manifested by increasedaccumulation of body fat. These lupus mice also have elevatedtriglycerides, similar to that seen in human SLE patients. A number ofstudies in murine SLE model have shown that dietary manipulations andrestrictions have an effect on the development on this life shorteningautoimmune disease. Several lines of evidence have supported a linkbetween adipose tissue and immunocompetent cells. For example, inobesity, excess adiposity is linked to impaired immune function. Studiesin rodents with genetic abnormality of leptin and leptin receptors,which result in obesity, revealed obesity-related changes in macrophagephagocytosis and the production of proinflammatory cytokines. These dataidentify an important link between obesity and regulation ofinflammatory and immune responses.

This experiment assesses the effect of the P-peptide on body fataccumulation in murine SLE model.

Briefly, female NZB/WF1 were obtained from Jackson Laboratories at 6weeks of age and housed locally for 2 weeks prior to initiation of thestudies. The study design comprised of four groups of 10 female NZB/WF1mice: one control and three experimental groups. The control group ofmice were not treated with 16 amino acid RHAMM peptide mimetic(P-peptide). First group of mice were given P-peptide (5 mg/kg), threetimes a week via the IP route. The treatment started at 8 weeks of ageand continued up to 28 weeks of age. The animals in the other twoexperimental groups were started on the P-peptide (5 mg/kg) at 16 and 24weeks of age to determine whether interference with P-peptide can arrestor reverse active weight gain. The treatment in these animals alsocontinued up to 28 weeks. The animals were assessed for weight gainduring the development of the disease at weekly intervals.

Results are shown in FIG. 45. Briefly, the control group of mice showeda trend of increase in body weight of approximately 5 g per month. Thetotal average increase after 20 weeks was 113 g. The group of mice thatwas treated from 8 weeks of age showed significant reduction in weightgain in comparison with the control group. The average increase of bodyweight in this group was 2 g per month, whereas total accumulation ofweight was 6 g after 20 weeks. Weight gain in mice in the other twoexperimental groups was identical to the control group until theinitiation of treatment. The body weigh in these mice showed decreasewithin the first week of the treatment, with the trend of furtherdecrease toward the levels observed in animals that were treated atearly stage of the disease (FIG. 45). The treatment with P-peptide didnot effect the weight gain in NOD mice, which served as a control forthis experiment.

In summary, the weight gain in mice that were treated at the early stageof disease (8 weeks) was similar to the weight gain in normal mice. Micethat were treated at later stages of disease showed not only arrest butreverse of weight gain that was similar to early stage treated mice.Thus, the P-peptide can be utilized as a therapeutic agent in thetreatment of obesity and obesity related diseases (e.g., diabetes andcardiovascular disease), as well as for diseases such as kidney fibrosisand lupus (SLE).

Example 27 Correlation Between RHAMM Levels and Cancer Cell Invasiveness

This experiment assesses the relationship between RHAMM expression andaggressiveness of cancer cell lines. RT-PCR was conducted as describedin the attached Wang et al., (Clinical Cancer Research, 4:567-576,1998). Western blot analyses was conducted as described above.

Results of these experiments are shown in FIG. 46. Briefly, the levelsof erk kinase correlated significantly with the levels of RHAMMexpression (r=0.21, p<0.007. Students “t” test). The cell lines H125.H157 and H226 are less invasive and aggressive than the H460 and MGH 7.As shown in the Figures. RHAMM expression is highest in the latter twocell lines. Of the two cell lines, the H460 is more invasive in matrigelassays than the MGH7 cell lines.

Based upon this experiment it is evident that the highest level of RHAMMexpression is observed in the most invasive lung cancer cell lines.

Example 28 Correlation Between Astrocytoma Cell Metastases and RHAMMExpression

Invasive astrocytoma cell lines (U87MG and U343MG-A), astrocytomabiopsies from patients, cervical tumor cell lines (HeLa) were extractedfor mRNA and analyzed for the presence of RHAMM using northern blots andRT-PCR as described by Sambrook et al. Results are shown in FIG. 47.Briefly, astrocytoma cell lines express approximately equal amounts ofRHAMM, as detected by Northern blot analysis. RT-PCR analysis show thatRHAMM is most highly expressed in high grade or invasive astrocytomas(FIG. 47B)

These results support that RHAMM is involved in the tumor invasion stepof neoplastic progression and this is consistent with its ability toregulate podosome formation, structures that permit release ofcollagenases that are required for cell invasion.

Example 29 Screening for Proteins that Regulate HA Transport in aTransitional Cell

A RHAMM induced cDNA expression library is obtained from mRNApopulations extracted from RHAMM transfected cells maintained in serumfree medium for 24 h. These culture conditions allow uptake of HA intothe cell cytoplasm but will not allow HA uptake into the cytoplasm ofnon-transfected cells unless a HA transport protein is expressed. ThecDNA library is used to infect COS or CHO cells which are then exposedto Texas red-labeled HA in the presence of cytochalasin D which inhibitsendocytic uptake of HA. Under these conditions cells would notordinarily take up HA into the cytoplasm, hence, HA uptake will dependon the expression of a HA transporter. Infected cells are brieflyexposed to streptomyces hyaluronidase to remove any Texas red labeled HAcoating the outside of the cell and then cells are sorted for positivefluorescence with FACS.

Cells that are positive are cloned and rescreened for HA uptake.Transfected genes encoding an HA transporter are then retrieved byRT-PCR of mRNA and sequenced. These genes are then transfected into10T1/2 cells which do not take up HA into the cytoplasm unless they areexposed to phorbol ester to activate protein kinase C. These cells arein turn assessed for uptake of Texas red-labeled HA into the cytoplasmand scored for altered growth factor sensitivity by techniquespreviously described herein.

The cDNA encoding a HA transporter is then cloned into an appropriateexpression vector that will permit expression and isolation of thetransporter protein. Appropriate vectors and expression systems are wellknown in the art. Antibodies are then be prepared against this protein.In addition, peptide regions instrumental in taking up HA (i.e., an HAbinding domains) are identified and peptides that mimic these sites areprepared for assessment of the ability to compete with HA transport orotherwise impact signaling pathways, podosome formation and/cellmotility which characterize transition stage cells.

Example 30

Identification of RHAMM Binding Proteins or Other Transition StageMolecules by use of RHAMM Overexpressing Cells Cultures

To identify proteins that are transiently regulated with RHAMM tocontrol cell activation, cDNA expression libraries obtained from the“CHIP” differential screen described above are used to establishlibraries expressing transition molecules that is capable of binding toa hyaladherin or other transition stage molecule. Several techniques areknown in the art for identifying an expressed binding partner. Theseinclude a trio hybrid phage display system and a two hybrid yeastexpression system. The two hybrid expression system is used to screenfor peptides or polypeptides that bind to RHAMM or other transitionmolecule, and the ability to actually bind the transition molecule isfurther characterized using a far Western assay system. Specific bindingregions of the RHAMM binding partners can be further identified usingthe functional regions of RHAMM exons and the regions of RHAMM known tobe involved in the transient phenotype through the ability to activateerk kinases as provided for example, in FIG. 48. Antibodies may be madeto the identified binding protein and assessed for the ability to affectcell motility or erk signaling cascades according as previouslydescribed.

One such protein herein designated as RABP for RHAMM Associated BindingProtein has been identified using this method by using a phage displaylibrary mentioned above to bind to the peptide regions of exon 4described as SEQ. ID NO: 17. A partial polypeptide and nucleic acidsequence for RABP is provided as SEQ. ID NO: 47 and 46. Antibody againstthis protein has been prepared and shown to be effective in inhibitingRHAMM activated podosome formation and signaling in RHAMM overexpressingcells. FIG. 49 shows the sequence for this novel RHAMM binding proteinwhich was determined to be a 60 kDa protein that binds to exon 4 ofRHAMM, and which is transiently present on the cell surface. Panel Ashows the partial sequence of RABP isolated by a two hybrid screen usingexon 4 of RHAMM. Panel B shows a Northern blot of RABP in transitionalcells. Panel C shows a Western blot of transitional cell lysateindicating that RABP occurs within a 60 kDa protein. Panel D is a FACSanalysis showing that RABP is present on the cell surface.

Other proteins regulating transition stage cells can be identified usingcell cultures characterized by the transition stage phenotypes describedfor LR21 cells provided herein. Briefly, transitional cell cultures thatoverexpress RHAMM at precisely the levels required for enhancingpodosome formation are grown to subconfluence (50-60%) in 10% FBS thenreleased from their substratum using a 0.15 median. PBS, non-enzymaticdisassociation medium. These cells are maintained in suspension indefined medium for 30 minutes and then plated for 24 hrs, at 5×15cells/ml on plasma fibronectin coated dishes which promotes podosomepositive, transitional phenotype. PolyA mRNA is isolated from thetransitional cells and a differential screen is conducted using a cellline that is plated onto plastic dishes so that podosomes are notproduced. RNA is placed into CHIPS for, differential screening and genesassociated with the transition phenotype are identified using CHIPprotein technology. Positive cDNA's are sequenced, cDNA libraries arescreened and RACE technology is used to obtain a full length cDNA.

The CHIP will contain cDNA's encoding proteins involved in thetransition stage phenotype that do not necessarily bind to HA but arenevertheless involved in regulating this stage. Hence, this method forobtaining transitional mRNA is useful for identifying other importantdominant acting proteins involved with the transitional stage ofresponse to injury processes. The CHIP screen can be used for proteinsthat bind to important podosome proteins such as CAS and cortactin, andfull length sequences of these can be obtained. The function of suchsequences may be analyzed for their effect on podosome formation bytransient transfection. The entire differentially screened mRNA can beused to transiently transfect cells to determine whether they can inducepodosome formation, using CAS or cortactin to detect podosomes asdescribed above. Particular sequences are identified by increasinglyrestricting the number of mRNAs included in a transfection group untilultimately restricting the size of the group to single mRNAs encodingsingle genes affecting the transition stage phenotype.

Example 31

Identification of Hyaladherins by Searching Databases for HyaluronanBinding Motifs

In addition to specific peptides such as those described in SEQ. ID NOS:1-10 that represent hyaladherins which bind to hyalauronic acid, avariant of additional polypeptides may be identified, generated andtested for use within the methods described herein. All such bindingmotifs are characterized by the presence of general amino acid motifsincluding staggered basic residues. These motifs can be more generallydescribed as BX7B (SEQ ID NO:28) where B is any basic amino acid and X7is any amino acid sequence of about seven residues but usually includingat least one hydrophobic amino acids or an additional basic amino acid.Most importantly however, none of the intervening X amino acids shouldbe acidic, as acidic amino acids appear to interfere with binding tohyaluronan, a negatively charged polymer. Peptides which arespecifically excluded from this motif include: BBXXBBBXXBB. KQKIKHVVKLK.KLKSQLVKRK. RYPISRPRKR, KNGRYSISR, RDGTRYVQKGEYR. RRRCGQKKK, RGTRSGSTR.RRRKKIQGRSKR. RKSYGKYQGR. KVGKSPPVR. KTFGKMKPR, RIKWSRVSK. KRTMRPTRR,KVGKSPPVR, or HREARSGKYK (SEQ ID Nos. 29-44 respectively). Theseexcluded peptides do not bind HA with the same high affinity as peptidesof the present invention which require are peptides that form an alphahelix. All motifs that bind to hyaluronan also preferably form strongalpha helices as predicted in secondary structure protein analysisprograms which further show that hyaluronan binding motifs contain atleast two basic amino acids aggregating along one plane of the helix.

Using this information, a search of the data bases for previouslyundetected hyaladherins can be made, searching first for theaforementioned motif then coupling this with analysis of the structuralrequirements again using protein prediction programs such as for exampleare available on the internet (e.g. GCG). Additional sequence candidatescan be found by searching appropriate databases of the technicalliterature such as Medline, Biosis, Chemical Abstracts and the like.Searches can then be made to determine which of the newly identifiedhyaladherins have previously been associated with disease and with theexpression of podosomes. Those that are present at the cell surface canthen be screened for their potential therapeutic use.

Example 32

Screening for Inhibitors of Podosome Formation

The present invention provides for novel cell lines that overexpressRHAMM and that produce enhanced formation of podosomes. These cell linesmay be utilized to screen for inhibitors of podosome formation.Concomitant with the formation of podosomes and development of atransient phenotype, cells release proteases that result in degradationof fibronectin; revealing a previously sequestered CS-1 sequence.Antibodies to this CS-1 sequence have been prepared. The presentlyprovided RHAMM overexpressing cell lines are coated on microsphere beadsin conjunction with plasma fibronectin to form an assay mixture which isincubated at 37° C. for 2-3 h. The aforementioned CS antibody conjugatedto a fluorochrome is added to this mixture causing a fluorescenceresponse indicative of fibronectin degradation which is in turnindicative of the formation of functional podosomes. Candidateinhibitors of podosome formation are identified by the ability to reducefluorescence in this assay and these candidates may be screened usingany of several high through-put screening systems known in the art.

Inhibitors to be screened include, but are not limited to antibodies, HAbinding peptides/polypeptides and RHAMM binding peptides/polypeptidesassociated with regulation, of the novel transitional stage cells asprovided in this invention. In addition, upon identification offunctional portions of newly discovered transitional proteins using themethods described herein, candidate inhibitors comprised of smallchemicals or peptide mimics of these functional portions can besynthesized according to methods known in the art. One set of peptidemimics includes for example, HA binding mimics.

Example 33 Humanized Antibodies that Inhibit Transitional MoleculeFunction

Humanized antibodies raised against transitional molecules identifiedabove (e.g. hyaladherins, RHAMM binding partners, transitional proteins)can be screened for their inhibition of specific cell signaling pathwaysinvolved in cell transition (inhibition of erk kinase activity, AP-1activity, MMP expression or specific transition states of the cell(e.g., podosome formation, cell migration, cell proliferation) influorescent screening assays. Cell lines over expressing specifictransition molecules will activate ERK kinases, c-fos expression, AP-1activity, MMP expression, and increased transitional states of the cellsuch as podosome formation resulting increased cell migration andproliferation.

Humanized antibodies to identified transition molecules such as RHAMMcan be screened for inhibition of the aforementioned cell signalingpathways, gene expression, podosome formation, and/or cell motility andproliferation. These studies will identify potent antibodies whichinhibit the transition of normal cells to diseased cells which can beutilized clinically in humans for the treatment and diagnosis ofdisease.

Example 34 Complementary Peptides and Peptide Mimics that Interfere withTransition Molecule Function

A variety of candidate peptides affecting transition state cells can bedetected and/or screened using the methods provided by the presentinvention. Candidate peptides include peptides generated from transitionmolecules provided in the present disclosure (such as the RHAMMpeptides), peptides of the transition stage which may further beidentified using the aforementioned methods, peptides that bind stronglyto active regions of transition molecules, or peptides that compete withbinding of transition molecules of specific ligands generated bystandard synthetic processes. Each of these can also be screened foreffects on the particular features of transition cells disclosed hereinincluding effects on specific signaling pathways (e.g., ERK activity,AP-1 activity,) gene expression (e.g. c-fos and MMP expression),podosome formation, cell motility and proliferation.

The structure of peptides effective in inhibiting transitionmolecule-induced processes can be determined by several methodsincluding standard structure function analyses of a proteins shown toinhibit podosome formation and/or by using the above screening methodsfor analyzing peptide sequences encoded by a gene shown to be involvedin podosome formation. Complementary peptides and peptide mimics can bedesigned based upon functional peptide motifs, particularly when aninhibitor peptide motiff is small (e.g. 10 amino acids or less). Suchpeptides and their mimics would be candidate molecules for therapeutictreatment of a variety of disease states dependent upon entry andpassage of cells through the transition stage phenotype taught by thepresent invention. Candidate molecules would be tested for efficacy byassay in animal models of disease or using cultured cells expressing atransitional stage cells.

Similar studies may be performed to screen small molecules for theirinhibition of transition molecule function and the progression of cellsfrom the normal to diseased state as described in the present invention.

Example 35

Diagnostics Method for Detecting HA. Hyaladherins and Injured Cells

1 Detection of Intracellular and Plasma HA

Serum and tissue levels of HA are valuable diagnostic markers ofarthritis and neoplasia. Thus, levels of HA in the serum are currentlyused to follow the course of osteoarthritis response to steroid therapy.Further, HA accumulation within colorectal and breast cancers isprognostic of a poor outcome. Because HA levels are enhanced followingmost forms of tissue injury, other conditions including restenosis, MS,Alzheimer's, stroke, myocardial infarction, sports injuries, burns andother inflammatory diseases would benefit from methods of detecting HA.In addition, HA increase in plasma is associated with a variety of otherdiseases, particularly rheumatoid arthritis and in tumors such asmesetheliomas and Wilm's tumors. Therefore testing of HA levels in serumor in biopsy tissue will be useful, alone or in combination with otherdisease markers for determination of a variety of disease conditions.

Currently, HA is routinely detected using fragments of HA bindingproteins such as the 60 kDa fragment of aggrecan or link protein. Theprocedures for purifying, these proteins is laborious and results areinconsistent making it difficult to routinely assess HA as a diagnosticparameter. The sensitivity of this technique is 5 pg HA in serum using,ELISA assays.

The present invention provides a method of similar sensitivity but whichis cheaper and more reliable. The method is based on using HA bindingpartners discovered using the techniques described above for detectingRHAMM binding partners. Using a phage display library to bind tobiotinylated HA permitted identification of five particular species ofHA binding motifs described in SEQ. ID NOS: 6-10. This was accomplishedby isolating phage that attached to HA which were further isolated,rescreened twice and recloned. The clones were then bound tobiotinylated HA-sepharose beads and only those phage that could bereleased with unlabeled HA were retrieved, recloned and sequenced. Fiveclones comprising the sequences identified above were detected. Thesesequences all bind to HA and are useful for detecting HA in serum andtissues in an assay described below.

An assay was developed based upon HA binding to these newly discoveredpeptide sequences. Synthetic peptides comprising these sequences weresynthesized with a linker arm of glycine-glycine-cysteine to which KLHwas covalently linked using EDAC. One to 200 μg of any one of thesepeptides were coated onto the surface of ELISA plates in phosphatebuffered saline for 1 h at room temperature. Plates were washed in PBSand then coated with 1 μg/ml of HA and washed with PBS and 0.1% triton.Texas red labeled peptide was then applied to the coated plate for 1 h.Serum samples and HA standard solutions were then applied to the platesand left on a mixer for 2 h. The plates were then washed and read in afluorescent ELISA plate reader at 545 μm. The amount of HA in thesamples was determined by comparison to the HA standards.

This assay has a similar sensitivity to previously described assaysusing, aggrecan but is more reproducible due to the standardizationpossible using peptide synthesis. This contrast to the more variableresults obtained using assays based on preparation of purified aggrecanfor which a reproducible reagent capable of binding to H-A is difficultto make.

These newly discovered peptides are also useful for detecting HA presentin tissue (e.g. biopsy tissue). In one example, frozen or paraffinembedded tissue sections are incubated with biotinylated peptides for 1h in sections that have been either exposed to hyaluronidase, used as acontrol, or to buffer alone. Sections are washed then developed withhorseradish peroxidase labeled streptavidin and sections are thenexamined for a positive reaction indicated by brown staining. Thisprocedure can be readily adapted for use in a kit as can the ELISA assayfor detecting HA in plasma.

2. Development of an Assay for Detection of Soluble Hyaladherins

The above mentioned HA binding peptides are also useful in an assay forsoluble hyaladherins. In this regard, an important aspect of the presentdisclosure is that the transition phenotype plays a heretoforeundisclosed role in many disease processes such as inflammatorydiseases, cancers, degenerative diseases and wound healing. In each caseHA will be shed during the podosome stage of a cell that typifies thetransitional phenotype. Therefore, the presence of a transitionalphenotype during the early stages of disease establishment may bedetected by assaying for the presence of hyalauronan or hyaladherinspresent in serum.

An assay for hyaladherins can be provided using the small peptides thatbind to HA as described herein before. In one example, these peptidescan be synthesized with an additional cysteine at the carboxy terminus.The peptides are then covalently linked to sepharose as per standardprocedures. The sepharose beads are incubated with biotinylated HA forone hour, then washed. The beads containing biotinylated HA are thenincubated for 1-2 hours at room temperature with an aliquot of sampleserum. Hyaladherins that are present within the serum will competewraith the peptide bound to sepharose for the biotinylated HA andtherefore the amount of biotin label remaining with the sepharose beadswill be inversely proportional to the amount of hyaladherins present inthe serum sample.

An alternative to the general hyaladherins assay mentioned above is aspecific hyaladherins assay for selected hyaladherins observed toincrease during a particular disease or cellular response as may bedetected using the screening, methods provided in the foregoingExamples. In this specific assay, monoclonal antibodies are preparedagainst the selected hyaladherins observed to increase during disease asdetected by these screening methods. The monoclonal antibodies are usedin a standard ELISA assay where antibodies are coated onto the ELISAwell, serum is added to this coating, washed and a second layer ofanti-hyaladherin will be layered on top. The top layer of antibody isdetected using a fluorochrome labeled secondary antibody and the amountof label quantified in an ELISA plate reader.

The presently described assays based upon use of HA binding peptideshyaladherins and antibodies thereto are readily adaptable for detectingother components associated with the transitional state such as HAtransporters or other proteins which may be detected using theaforementioned screening systems.

Example 36 RHAMM Regulates Prostate Cancer Progression

This experiment investigates whether functional expression of the HAreceptor RHAMM is required for enhancement of CaP cell motility andinvasion in vitro.

Briefly, Dunning CaP cell lines (AT-1, MatLyLu) were grown in DMEMmedium supplemented with 10% FBS at 37° C. in a humidified atmospherecontaining 5% CO₂. All cell lines were passaged every 3-4 days uponreaching confluency.

A. Immunofluorescence.

Cells were seeded sparsely on glass coverslip and incubated in growthmedia for 24 h. cells were then fixed with 3% paraformaldehyde andpermeabilized with 0.2% triton X-100. RHAMM was visualized by indirectimmunofluorescence using a polyclonal antibody to the C-terminus (Zram2.3, 1:100) and Texas red conjugated donkey anti-rabbit antibody(1:100). Images are obtained using a Zeiss laser scanning confocalmicroscope.

B. Western Blotting.

Cells were also grown to 50-60% confluency were lysed using RIPA buffer.Equal amounts of total cell protein were loaded onto a 10% SDS-PAGE (el.RHAMM was probed using a polyclonal antibody to the C-terminus (Zram,1:1000) and HRP-conjugated goat anti-rabbit antibody (1:5000). RHAMM wasvisualized by chemiluminescence.

C. Cell Motility.

Cell were seeded sparsely and crown in 25 cm² flasks overnight.Serum-free medium was used for the experiments. Random cell motility ofcells untreated, or treated with either RHAMM polyclonal antibody (Re4)or peptide mimicking the HA-binding domain over two hours was visualizedby videomicroscopy. Cell motility tracks were analyzed using a NorthernExposure software. Statistical analysis was performed on 100 cells perfield and statistical significance was determined using unpaired Studentt-test.

D. Cell Invasion.

Cell were grown to confluency in growth media, detached, and equalnumber of cells were seeded in 24-well Matrigel invasion chambers. Cellswere left untreated with RHAMM peptide and allowed to invade for 24 h.For statistical analysis, 5 high-power fields(400×) were counted for thenumber of cells that invaded through the membrane. Statisticalsignificance was determined using unpaired Student t-test.

E. MMP Activity.

Cells were grown to confluency in growth media, detached and equalnumber of cells were seeded in 6-well plates uncoated or coated with 50%Matrigel in media. Cells were allowed to adhere for 1 h to thesubstrate, and then treated with the peptide mimicking the HA-bindingdomain of RHAMM (100 μg/ml) for 24 h in serum-free media. The activityof MMP secreted into the media was determined by zymography using 8%SDS-PAGE.

Thus, RHAMM is preferentially expressed in more motile/invasive andmetastatic CaP cells. Blocking RHAMM function significantly andpreferentially reduces motility, invasion, and MMP activity in highlymetastatic CaP cells.

Example 37

Treatment and/or Prevention of Diabetes Mellitus

The purpose of these experiments was to evaluate the RHAMM (P-16)peptide on the treatment of diabetes in the non-obese diabetic (NOD)mouse model. NOD mouse is a model of human type I diabetes mellitus,which is characterized by a cell-mediated autoimmune process resultingin spontaneous diabetes (see, e.g. Zaho et al., Lithium (1991), 2(4),227-34; see also, The Jackson Laboratory). Studies have shown that themajor populations of cells infiltrating the islets of Langerhans in theearly stage of insulitis in NOD mice are T cells and macrophages.

There are different colonies of NOD mice and there can be somevariability between the colonies. The colony used develops the diseasebetween 15-20 weeks of age and there is a 70-80% incidence of diabetesmellitus. The mice treated were divided into two groups of 10 animals;the first group being treated with P-16 peptide and the other groupcomprising of the control group, which was treated with saline. Once theNOD mice were 5 weeks old, the P-16 peptide was injected three times aweek interperitoneally at a dose of 5 mg/kg for 23 weeks. The untreatedmice and five mice from the treated group were sacrificed at 28 weeks ofage. The remaining five mice from the treated group were taken off thepeptide treatment at 28 weeks of age and were assessed for the diseaseafter 16 weeks.

As shown in FIG. 51, the incidence of diabetes measured by blood glucoselevel in untreated NOD mice was 70% whereas the incidence in the treatedmice was 20% The untreated mice also had a higher incidence of abnormalurine glucose level, 80%, compared to 0% in the treated mice (FIG. 52).Further, when examining water consumption associated with diabetes,water consumption increased significantly in untreated animals with theonset of diabetes around week 12 to 13 (FIG. 53). In contrast, the waterconsumption did not change in animals treated with P-16. These dataclearly so demonstrate that P-16 peptide inhibited the incidence ofdiabetes.

The treated mice that had the treatment stopped at 28 weeks have notdeveloped any signs of the disease after 16 weeks. They looked healthyand did not show presence of polydipsia or urinary glucose.

In NOD mice, there was an increase in kidney weight due to renalhypertrophy that is associated with the onset and progression ofdiabetic symptoms. As shown in FIG. 54, treatment with the P-16completely inhibited the increase in kidney weight, presumably byinhibiting glomerulosclerosis.

The histological analysis of pancreatic tissue showed that treated micehad more intact pancreatic islets than the untreated animals andsignificantly smaller inflammation of the islets with inflammatorycells.

Presented results clearly show that RHAMM (P-16) peptide administrationpotently prevents the development of diabetes and associatedcomplications in the NOD model of Type I diabetes mellitus in theabsence of any toxicity. The diabetes-sparing effect is probably due tothe inhibition of the destruction of beta cells in the pancreaticislets. The effectiveness of RHAMM (P-16) peptide administration toinduce long-term inhibition of disease was demonstrated by the negativeresults of urinary glucose and polydipsia of 16 weeks post-peptidetreatment NOD mice.

These results indicate that RHAMM and its major ligand HA associatefunctionally with autoimmune insulitis leading to IDDM, and that byusing specific RHAMM peptides they can serve as potential therapeutictargets.

These findings also show that the RHAMM peptides, peptide mimetics,antibodies and potential HA binding peptides can be used as an effectivemethod for preventing and/or treating diabetes mellitus by interferingwith the penetration of the inflammatory cells into the islets anddestructive invasion of the islets.

From the foregoing, it will be appreciated that, although specificembodiments of the invention have been described herein for purposes ofillustration, various modifications may be made without deviating fromthe spirit and scope of the invention.

1. A method for treating a patient with a inflammatory neurologicaldisorder, comprising administering to a patient a compound selected fromthe group consisting of (a) a polypeptide comprising the amino acidsequence BX7B which binds HA; (b) an antibody which binds one of domainsD1, D2, D3, D4, or, D5 of RHAMM; (c) a polypeptide fragment whichencodes a D1, D2, D3, D4, or, D5 domain of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that said inflammatoryneurological disorder is treated.
 2. The method according to claim 1wherein said inflammatory neurological disorder is Parkinsons disease.3. The method according to claim 1 wherein said inflammatoryneurological disorder is Alzheimer disease
 4. A method for treating apatient with arthritis, comprising administering to a patient a compoundselected from the group consisting of (a) a polypeptide comprising theamino acid sequence BX7B which binds HA; (b) an antibody which binds oneof domains D1, D2, D3, D4, or, D5 of RHAMM; (c) a polypeptide fragmentwhich encodes a D1, D2, D3, D4, or, D5 domain of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that said arthritis istreated.
 5. The method according to claim 4 wherein said arthritis isrheumatoid arthritis.
 6. The method according to claim 4 wherein saidarthritis is osteoarthritis.
 7. A method for treating a patient withmultiple sclerosis, comprising administering to a patient a compoundselected from the group consisting of (a) a polypeptide comprising theamino acid sequence BX7B which binds HA; (b) an antibody which binds oneof domains D1, D2, D3, D4, or, D5 of RHAMM; (c) a polypeptide fragmentwhich encodes a D1, D2, D3, D4, or, D5 domain of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that said multiple sclerosisis treated.
 8. A method for treating a patient with inflammatorydermatosis, comprising administering to a patient a compound selectedfrom the group consisting of (a) a polypeptide comprising the amino acidsequence BX7B which binds HA; (b) an antibody which binds one of domainsD1, D2, D3, D4, or, D5 of RHAMM; (c) a polypeptide fragment whichencodes a D1, D2, D3, D4, or, D5 domain of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that said inflammatorydermatitis is treated.
 9. The method according to claim 8 wherein saidinflammatory dermatosis is psoriasis.
 10. A method for treating apatient with inflammatory, bowel disease, comprising administering to apatient a compound selected from the group consisting of (a) apolypeptide comprising the amino acid sequence BX7B which binds HA; (b)an antibody which binds one of domains D1, D2, D3, D4, or, D5 of RHAMM;(c) a polypeptide fragment which encodes a D1, D2, D3, D4, or, D5 domainof RHAMM, and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatsaid inflammatory bowel disease is treated.
 11. A method for treatingwounds, comprising administering to a patient a compound selected fromthe group consisting of (a) a polypeptide comprising the amino acidsequence BX7B which binds HA; (b) an antibody which binds one of domainsD1, D2, D3, D4, or, D5 of RHAMM; (c) a polypeptide fragment whichencodes a D1, D2, D3, D4, or, D5 domain of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that said wound is treated.12. The method according to claim 11 wherein said wound is a surgicalexcision.
 13. The method according to claim 11 wherein said method isutilized to prevent a surgical adhesion.
 14. The method according toclaim 11 wherein said method is utilized to prevent a scar.
 15. A methodfor treating stenosis or restenosis, comprising administering to apatient a compound selected from the group consisting of (a) apolypeptide comprising the amino acid sequence BX7B which binds HA; (b)an antibody which binds one of domains D1, D2 D3, D4, or, D5 of RHAMM;(c) a polypeptide fragment which encodes a D1, D2, D3, D4, or, D5 domainof RHAMM; and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatsaid stenosis or restenosis is treated.
 16. The method according toclaim 15, wherein said compound is administered through a ballooncatheter.
 17. The method according to claim 15 wherein said compound isapplied to a stent, which is placed in said patient.
 18. The methodaccording to claim 15 wherein said compound is administered to theoutside to the vessel to be treated.
 19. A method for treating cancer,comprising administering to a patient a compound selected from the groupconsisting of (a) a polypeptide comprising the amino acid sequence BX7Bwhich binds HA; (b) an antibody which binds one of domains D1, D2, D3,D4, or, D5 of RHAMM; (c) a polypeptide fragment which encodes a D1, D2,D3, D4, or, D5 domain of RHAMM; and (d) a gene delivery vector whichexpresses antisense RHAMM, or, delivers and expresses any one of (a),(b), or (c), such that said cancer is treated.
 20. A method for treatingkidney fibrosis, comprising administering to a patient a compoundselected from the group consisting of (a) a polypeptide comprising theamino acid sequence BX7B which binds HA; (b) an antibody which binds oneof domains D1, D2, D3, D4, or, D5 of RHAMM; (c) a polypeptide fragmentwhich encodes a D1, D2, D3, D4, or, D5 domain of RHAMM; and (d) a genedelivery vector which expresses antisense RHAMM, or, delivers andexpresses any one of (a), (b), or (c), such that said kidney fibrosis istreated.
 21. A method for treating inflammatory lung disease, comprisingadministering to a patient a compound selected from the group consistingof (a) a polypeptide comprising the amino acid sequence BX7B which bindsHA; (b) an antibody which binds one of domains D1, D2, D3, D4, or, D5 ofRHAMM; (c) a polypeptide fragment which encodes a D1, D2, D3, D4, or, D5domain of RHAMM; and (d) a gene delivery vector which expressesantisense RHAMM, or, delivers and expresses any one of (a), (b), or (c),such that said inflammatory lung disease is treated.
 22. The methodaccording to claim 21 wherein said inflammatory lung disease isemphysema.
 23. The method according to claim 21 wherein saidinflammatory lung disease is asthma.
 24. The method according to claim21 wherein said inflammatory lung disease is cystic fibrosis.
 25. Amethod for treating obesity and obesity related diseases, comprisingadministering to a patient a compound selected from the group consistingof (a) a polypeptide comprising the amino acid sequence BX7B which bindsHA; (b) an antibody which binds one of domains D1, D2, D3, D4, or, D5 ofRHAMM; (c) a polypeptide fragment which encodes a D1, D2, D3, D4, or, D5domain of RHAMM; and (d) a gene delivery vector which expressesantisense RHAMM, or, delivers and expresses any one of (a), (b), or (c),such that said obesity and obesity-related diseases are treated.
 26. Amethod for treating lupus, comprising administering to a patient acompound selected from the group consisting of (a) a polypeptidecomprising the amino acid sequence BX7B which binds HA; (b) an antibodywhich binds one of domains D1, D2, D3, D4, or, D5 of RHAMM; (c) apolypeptide fragment which encodes a D1, D2, D3, D4, or, D5 domain ofRHAMM; and (d) a gene delivery vector which expresses antisense RHAMM,or, delivers and expresses any one of (a), (b), or (c), such that saidlupus is treated.
 27. A method for treating cardiovascular disease,comprising administering to a patient a compound selected from the groupconsisting of (a) a polypeptide comprising the amino acid sequence BX7Bwhich binds HA; (b) an antibody which binds one of domains D1, D2, D3,D4, or, D5 of RHAMM; (c) a polypeptide fragment which encodes a D1, D2,D3, D4, or, D5 domain of RHAMM; and (d) a gene delivery vector whichexpresses antisense RHAMM, or, delivers and expresses any one of (a),(b), or (c), such that said cardiovascular disease is treated.
 28. Themethod according to claim 27 wherein said cardiovascular disease isatherosclerosis.
 29. An antibody which binds to any one of domains D1,D2, D3, D4, or, D5 of RHAMM.
 30. The antibody according to claim 29wherein said RHAMM is human RHAMM.
 31. The antibody according to claim29 wherein said antibody is a monoclonal antibody.
 32. The antibodyaccording to claim 31 wherein said monoclonal antibody is a humanmonoclonal antibody.
 33. The antibody according to claim 31 wherein saidmonoclonal antibody is an Fab fragment of an antibody.
 34. A polypeptidefragment comprising all or a portion of domains D1, D2, D3, D4, or, D5of RHAMM, wherein said polypeptide is less than 73 kD molecular weight.35. The polypeptide fragment according to claim 34, wherein saidpolypeptide is less than 100 amino acids in length.
 36. The polypeptidefragment according to claim 34 wherein said polypeptide fragment is lessthan 75 amino acids in length.
 37. A method for treating or preventingdiabetes mellitus, comprising administering to a patient a compoundselected from the group consisting of (a) a polypeptide comprising theamino acid sequence BX7B (SEQ ID NO:28); (b) an antibody which binds oneof domains D1, D2, D3, D4, or D5 of RHAMM; (c) a peptide of less than 95kD or 73 kd, comprising all or a portion of domains D1, D2, D3, D4, or,D5 of RHAMM; and (d) a gene delivery vector which expresses antisenseRHAMM, or, delivers and expresses any one of (a), (b), or (c), such thatthe disease is treated.